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Sample records for quantitative phosphoproteomics dissection

  1. Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

    PubMed Central

    Jacobs, Kris; Klammer, Martin; Jordan, Nicole; Elschenbroich, Sarah; Parade, Marc; Jacoby, Edgar; Linders, Joannes T. M.; Brehmer, Dirk; Cools, Jan; Daub, Henrik

    2016-01-01

    The four members of the epidermal growth factor receptor (EGFR/ERBB) family form homo- and heterodimers which mediate ligand-specific regulation of many key cellular processes in normal and cancer tissues. While signaling through the EGFR has been extensively studied on the molecular level, signal transduction through ERBB3/ERBB4 heterodimers is less well understood. Here, we generated isogenic mouse Ba/F3 cells that express full-length and functional membrane-integrated ERBB3 and ERBB4 or ERBB4 alone, to serve as a defined cellular model for biological and phosphoproteomics analysis of ERBB3/ERBB4 signaling. ERBB3 co-expression significantly enhanced Ba/F3 cell proliferation upon neuregulin-1 (NRG1) treatment. For comprehensive signaling studies we performed quantitative mass spectrometry (MS) experiments to compare the basal ERBB3/ERBB4 cell phosphoproteome to NRG1 treatment of ERBB3/ERBB4 and ERBB4 cells. We employed a workflow comprising differential isotope labeling with mTRAQ reagents followed by chromatographic peptide separation and final phosphopeptide enrichment prior to MS analysis. Overall, we identified 9686 phosphorylation sites which could be confidently localized to specific residues. Statistical analysis of three replicate experiments revealed 492 phosphorylation sites which were significantly changed in NRG1-treated ERBB3/ERBB4 cells. Bioinformatics data analysis recapitulated regulation of mitogen-activated protein kinase and Akt pathways, but also indicated signaling links to cytoskeletal functions and nuclear biology. Comparative assessment of NRG1-stimulated ERBB4 Ba/F3 cells revealed that ERBB3 did not trigger defined signaling pathways but more broadly enhanced phosphoproteome regulation in cells expressing both receptors. In conclusion, our data provide the first global picture of ERBB3/ERBB4 signaling and provide numerous potential starting points for further mechanistic studies. PMID:26745281

  2. Quantitative Phosphoproteomics Reveals Extensive Cellular Reprogramming During HIV-1 Entry

    PubMed Central

    Wojcechowskyj, Jason A.; Didigu, Chuka A.; Lee, Jessica Y.; Parrish, Nicholas F.; Sinha, Rohini; Hahn, Beatrice H.; Bushman, Frederic D.; Jensen, Shane T.; Seeholzer, Steven H.; Doms, Robert W.

    2014-01-01

    SUMMARY Receptor engagement by HIV-1 during host cell entry activates signaling pathways that can reprogram the cell for optimal viral replication. To obtain a global view of the signaling events induced during HIV-1 entry, we conducted a quantitative phosphoproteomics screen of primary human CD4+ T cell after infection with an HIV-1 strain that engages the receptors CD4 and CXCR4. We quantified 1,757 phosphorylation sites with high stringency. The abundance of 239 phosphorylation sites from 175 genes, including several proteins in pathways known to be impacted by HIV-receptor binding, changed significantly within a minute after HIV-1 exposure. Several previously uncharacterized HIV-1 host factors were also identified and confirmed through RNAi depletion studies. Surprisingly, 5 serine/arginine-rich (SR)-proteins involved in mRNA splicing, including the splicing factor SRm300 (SRRM2) were differentially phosophorylated. Mechanistic studies with SRRM2 suggest that HIV-1 modulates host cell alternative splicing machinery during entry in order to facilitate virus replication and release. PMID:23684312

  3. Quantitative phosphoproteomics by mass spectrometry: past, present, and future.

    PubMed

    Nita-Lazar, Aleksandra; Saito-Benz, Hideshiro; White, Forest M

    2008-11-01

    Protein phosphorylation-mediated signaling networks regulate much of the cellular response to external stimuli, and dysregulation in these networks has been linked to multiple disease states. Significant advancements have been made over the past decade to enable the analysis and quantification of cellular protein phosphorylation events, but comprehensive analysis of the phosphoproteome is still lacking, as is the ability to monitor signaling at the network level while comprehending the biological implications of each phosphorylation site. In this review we highlight many of the technological advances over the past decade and describe some of the latest applications of these tools to uncover signaling networks in a variety of biological settings. We finish with a concise discussion of the future of the field, including additional advances that are required to link protein phosphorylation analysis with biological insight.

  4. Cost-effective isobaric tagging for quantitative phosphoproteomics using DiART reagents.

    PubMed

    Ramsubramaniam, Nikhil; Tao, Feng; Li, Shuwei; Marten, Mark R

    2013-12-01

    We describe the use of an isobaric tagging reagent, Deuterium isobaric Amine Reactive Tag (DiART), for quantitative phosphoproteomic experiments. Using DiART tagged custom mixtures of two phosphorylated peptides from alpha casein and their non-phosphorylated counterparts, we demonstrate the compatibility of DiART with TiO2 affinity purification of phosphorylated peptides. Comparison of theoretical vs. experimental reporter ion ratios reveals accurate quantification of phosphorylated peptides over a dynamic range of more than 15-fold. Using DiART labelling and TiO2 enrichment (DiART-TiO2) with large quantities of proteins (8 mg) from the cell lysate of model fungus Aspergillus nidulans, we quantified 744 unique phosphopeptides. Overlap of median values of TiO2 enriched phosphopeptides with theoretical values indicates accurate trends. Altogether these findings confirm the feasibility of performing quantitative phosphoproteomic experiments in a cost-effective manner using isobaric tagging reagents, DiART.

  5. Quantitative Phosphoproteomics Revealed Glucose-Stimulated Responses of Islet Associated with Insulin Secretion.

    PubMed

    Li, Jiaming; Li, Qingrun; Tang, Jiashu; Xia, Fangying; Wu, Jiarui; Zeng, Rong

    2015-11-01

    As central tissue of glucose homeostasis, islet has been an important focus of diabetes research. Phosphorylation plays pivotal roles in islet function, especially in islet glucose-stimulated insulin secretion. A systematic view on how phosphorylation networks were coordinately regulated in this process remains lacking, partially due to the limited amount of islets from an individual for a phosphoproteomic analysis. Here we optimized the in-tip and best-ratio phosphopeptide enrichment strategy and a SILAC-based workflow for processing rat islet samples. With limited islet lysates from each individual rat (20-47 μg), we identified 8539 phosphosites on 2487 proteins. Subsequent quantitative analyses uncovered that short-term (30 min) high glucose stimulation induced coordinate responses of islet phosphoproteome on multiple biological levels, including insulin secretion related pathways, cytoskeleton dynamics, protein processing in ER and Golgi, transcription and translation, and so on. Furthermore, three glucose-responsive phosphosites (Prkar1a pT75pS77 and Tagln2 pS163) from the data set were proved to be correlated with insulin secretion. Overall, we initially gave an in-depth map of islet phosphoproteome regulated by glucose on individual rat level. This was a significant addition to our knowledge about how phosphorylation networks responded in insulin secretion. Also, the list of changed phosphosites was a valuable resource for molecular researchers in diabetes field. PMID:26437020

  6. Quantitative Phosphoproteomics Revealed Glucose-Stimulated Responses of Islet Associated with Insulin Secretion.

    PubMed

    Li, Jiaming; Li, Qingrun; Tang, Jiashu; Xia, Fangying; Wu, Jiarui; Zeng, Rong

    2015-11-01

    As central tissue of glucose homeostasis, islet has been an important focus of diabetes research. Phosphorylation plays pivotal roles in islet function, especially in islet glucose-stimulated insulin secretion. A systematic view on how phosphorylation networks were coordinately regulated in this process remains lacking, partially due to the limited amount of islets from an individual for a phosphoproteomic analysis. Here we optimized the in-tip and best-ratio phosphopeptide enrichment strategy and a SILAC-based workflow for processing rat islet samples. With limited islet lysates from each individual rat (20-47 μg), we identified 8539 phosphosites on 2487 proteins. Subsequent quantitative analyses uncovered that short-term (30 min) high glucose stimulation induced coordinate responses of islet phosphoproteome on multiple biological levels, including insulin secretion related pathways, cytoskeleton dynamics, protein processing in ER and Golgi, transcription and translation, and so on. Furthermore, three glucose-responsive phosphosites (Prkar1a pT75pS77 and Tagln2 pS163) from the data set were proved to be correlated with insulin secretion. Overall, we initially gave an in-depth map of islet phosphoproteome regulated by glucose on individual rat level. This was a significant addition to our knowledge about how phosphorylation networks responded in insulin secretion. Also, the list of changed phosphosites was a valuable resource for molecular researchers in diabetes field.

  7. Quantitative phospho-proteomics reveals the Plasmodium merozoite triggers pre-invasion host kinase modification of the red cell cytoskeleton.

    PubMed

    Zuccala, Elizabeth S; Satchwell, Timothy J; Angrisano, Fiona; Tan, Yan Hong; Wilson, Marieangela C; Heesom, Kate J; Baum, Jake

    2016-01-01

    The invasive blood-stage malaria parasite - the merozoite - induces rapid morphological changes to the target erythrocyte during entry. However, evidence for active molecular changes in the host cell that accompany merozoite invasion is lacking. Here, we use invasion inhibition assays, erythrocyte resealing and high-definition imaging to explore red cell responses during invasion. We show that although merozoite entry does not involve erythrocyte actin reorganisation, it does require ATP to complete the process. Towards dissecting the ATP requirement, we present an in depth quantitative phospho-proteomic analysis of the erythrocyte during each stage of invasion. Specifically, we demonstrate extensive increased phosphorylation of erythrocyte proteins on merozoite attachment, including modification of the cytoskeletal proteins beta-spectrin and PIEZO1. The association with merozoite contact but not active entry demonstrates that parasite-dependent phosphorylation is mediated by host-cell kinase activity. This provides the first evidence that the erythrocyte is stimulated to respond to early invasion events through molecular changes in its membrane architecture. PMID:26830761

  8. Quantitative phospho-proteomics reveals the Plasmodium merozoite triggers pre-invasion host kinase modification of the red cell cytoskeleton

    PubMed Central

    Zuccala, Elizabeth S.; Satchwell, Timothy J.; Angrisano, Fiona; Tan, Yan Hong; Wilson, Marieangela C.; Heesom, Kate J.; Baum, Jake

    2016-01-01

    The invasive blood-stage malaria parasite – the merozoite – induces rapid morphological changes to the target erythrocyte during entry. However, evidence for active molecular changes in the host cell that accompany merozoite invasion is lacking. Here, we use invasion inhibition assays, erythrocyte resealing and high-definition imaging to explore red cell responses during invasion. We show that although merozoite entry does not involve erythrocyte actin reorganisation, it does require ATP to complete the process. Towards dissecting the ATP requirement, we present an in depth quantitative phospho-proteomic analysis of the erythrocyte during each stage of invasion. Specifically, we demonstrate extensive increased phosphorylation of erythrocyte proteins on merozoite attachment, including modification of the cytoskeletal proteins beta-spectrin and PIEZO1. The association with merozoite contact but not active entry demonstrates that parasite-dependent phosphorylation is mediated by host-cell kinase activity. This provides the first evidence that the erythrocyte is stimulated to respond to early invasion events through molecular changes in its membrane architecture. PMID:26830761

  9. Quantitative analysis of the TNF-α-induced phosphoproteome reveals AEG-1/MTDH/LYRIC as an IKKβ substrate

    PubMed Central

    Krishnan, Ramesh K.; Nolte, Hendrik; Sun, Tianliang; Kaur, Harmandeep; Sreenivasan, Krishnamoorthy; Looso, Mario; Offermanns, Stefan; Krüger, Marcus; Swiercz, Jakub M.

    2015-01-01

    The inhibitor of the nuclear factor-κB (IκB) kinase (IKK) complex is a key regulator of the canonical NF-κB signalling cascade and is crucial for fundamental cellular functions, including stress and immune responses. The majority of IKK complex functions are attributed to NF-κB activation; however, there is increasing evidence for NF-κB pathway-independent signalling. Here we combine quantitative mass spectrometry with random forest bioinformatics to dissect the TNF-α-IKKβ-induced phosphoproteome in MCF-7 breast cancer cells. In total, we identify over 20,000 phosphorylation sites, of which ∼1% are regulated up on TNF-α stimulation. We identify various potential novel IKKβ substrates including kinases and regulators of cellular trafficking. Moreover, we show that one of the candidates, AEG-1/MTDH/LYRIC, is directly phosphorylated by IKKβ on serine 298. We provide evidence that IKKβ-mediated AEG-1 phosphorylation is essential for IκBα degradation as well as NF-κB-dependent gene expression and cell proliferation, which correlate with cancer patient survival in vivo. PMID:25849741

  10. Quantitative phospho-proteomics reveals the Plasmodium merozoite triggers pre-invasion host kinase modification of the red cell cytoskeleton.

    PubMed

    Zuccala, Elizabeth S; Satchwell, Timothy J; Angrisano, Fiona; Tan, Yan Hong; Wilson, Marieangela C; Heesom, Kate J; Baum, Jake

    2016-02-02

    The invasive blood-stage malaria parasite - the merozoite - induces rapid morphological changes to the target erythrocyte during entry. However, evidence for active molecular changes in the host cell that accompany merozoite invasion is lacking. Here, we use invasion inhibition assays, erythrocyte resealing and high-definition imaging to explore red cell responses during invasion. We show that although merozoite entry does not involve erythrocyte actin reorganisation, it does require ATP to complete the process. Towards dissecting the ATP requirement, we present an in depth quantitative phospho-proteomic analysis of the erythrocyte during each stage of invasion. Specifically, we demonstrate extensive increased phosphorylation of erythrocyte proteins on merozoite attachment, including modification of the cytoskeletal proteins beta-spectrin and PIEZO1. The association with merozoite contact but not active entry demonstrates that parasite-dependent phosphorylation is mediated by host-cell kinase activity. This provides the first evidence that the erythrocyte is stimulated to respond to early invasion events through molecular changes in its membrane architecture.

  11. Quantitative phosphoproteomic analysis of early seed development in rice (Oryza sativa L.).

    PubMed

    Qiu, Jiehua; Hou, Yuxuan; Tong, Xiaohong; Wang, Yifeng; Lin, Haiyan; Liu, Qing; Zhang, Wen; Li, Zhiyong; Nallamilli, Babi R; Zhang, Jian

    2016-02-01

    Rice (Oryza sativa L.) seed serves as a major food source for over half of the global population. Though it has been long recognized that phosphorylation plays an essential role in rice seed development, the phosphorylation events and dynamics in this process remain largely unknown so far. Here, we report the first large scale identification of rice seed phosphoproteins and phosphosites by using a quantitative phosphoproteomic approach. Thorough proteomic studies in pistils and seeds at 3, 7 days after pollination resulted in the successful identification of 3885, 4313 and 4135 phosphopeptides respectively. A total of 2487 proteins were differentially phosphorylated among the three stages, including Kip related protein 1, Rice basic leucine zipper factor 1, Rice prolamin box binding factor and numerous other master regulators of rice seed development. Moreover, differentially phosphorylated proteins may be extensively involved in the biosynthesis and signaling pathways of phytohormones such as auxin, gibberellin, abscisic acid and brassinosteroid. Our results strongly indicated that protein phosphorylation is a key mechanism regulating cell proliferation and enlargement, phytohormone biosynthesis and signaling, grain filling and grain quality during rice seed development. Overall, the current study enhanced our understanding of the rice phosphoproteome and shed novel insight into the regulatory mechanism of rice seed development.

  12. A quantitative map of the liver mitochondrial phosphoproteome reveals post-translational control of ketogenesis

    PubMed Central

    Grimsrud, Paul A.; Carson, Joshua J.; Hebert, Alex S.; Hubler, Shane L.; Niemi, Natalie M.; Bailey, Derek J.; Jochem, Adam; Stapleton, Donald S.; Keller, Mark P.; Westphall, Michael S.; Yandell, Brian S.; Attie, Alan D.; Coon, Joshua J.; Pagliarini, David J.

    2012-01-01

    Summary Mitochondria are dynamic organelles that play a central role in a diverse array of metabolic processes. Elucidating mitochondrial adaptations to changing metabolic demands and the pathogenic alterations that underlie metabolic disorders represent principal challenges in cell biology. Here, we performed multiplexed quantitative mass spectrometry-based proteomics to chart the remodeling of the mouse liver mitochondrial proteome and phosphoproteome during both acute and chronic physiological transformations in more than 50 mice. Our analyses reveal that reversible phosphorylation is widespread in mitochondria, and is a key mechanism for regulating ketogenesis during the onset of obesity and type 2 diabetes. Specifically, we have demonstrated that phosphorylation of a conserved serine on Hmgcs2 (S456) significantly enhances its catalytic activity in response to increased ketogenic demand. Collectively, our work describes the plasticity of this organelle at high resolution and provides a framework for investigating the roles of proteome restructuring and reversible phosphorylation in mitochondrial adaptation. PMID:23140645

  13. Quantitative Analysis of Human Pluripotency and Neural Specification by In-Depth (Phospho)Proteomic Profiling.

    PubMed

    Singec, Ilyas; Crain, Andrew M; Hou, Junjie; Tobe, Brian T D; Talantova, Maria; Winquist, Alicia A; Doctor, Kutbuddin S; Choy, Jennifer; Huang, Xiayu; La Monaca, Esther; Horn, David M; Wolf, Dieter A; Lipton, Stuart A; Gutierrez, Gustavo J; Brill, Laurence M; Snyder, Evan Y

    2016-09-13

    Controlled differentiation of human embryonic stem cells (hESCs) can be utilized for precise analysis of cell type identities during early development. We established a highly efficient neural induction strategy and an improved analytical platform, and determined proteomic and phosphoproteomic profiles of hESCs and their specified multipotent neural stem cell derivatives (hNSCs). This quantitative dataset (nearly 13,000 proteins and 60,000 phosphorylation sites) provides unique molecular insights into pluripotency and neural lineage entry. Systems-level comparative analysis of proteins (e.g., transcription factors, epigenetic regulators, kinase families), phosphorylation sites, and numerous biological pathways allowed the identification of distinct signatures in pluripotent and multipotent cells. Furthermore, as predicted by the dataset, we functionally validated an autocrine/paracrine mechanism by demonstrating that the secreted protein midkine is a regulator of neural specification. This resource is freely available to the scientific community, including a searchable website, PluriProt. PMID:27569059

  14. Phosphoproteome analysis.

    PubMed

    Raggiaschi, Roberto; Gotta, Stefano; Terstappen, Georg C

    2005-01-01

    Protein phosphorylation is directly or indirectly involved in all important cellular events. The understanding of its regulatory role requires the discovery of the proteins involved in these processes and how, where and when protein phosphorylation takes place. Investigation of the phosphoproteome of a cell is becoming feasible today although it still represents a very difficult task especially if quantitative comparisons have to be made. Several different experimental strategies can be employed to explore phosphoproteomes and this review will cover the most important ones such as incorporation of radiolabeled phosphate into proteins, application of specific antibodies against phosphorylated residues and direct staining of phosphorylated proteins in polyacrylamide gels. Moreover, methods to enrich phosphorylated proteins such as affinity chromatography (IMAC) and immunoprecipitation as well as mass spectrometry for identification of phosphorylated peptides and phosphorylation sites are also described.

  15. Quantitative proteomics and phosphoproteomics on serial tumor biopsies from a sorafenib-treated HCC patient

    PubMed Central

    Dazert, Eva; Colombi, Marco; Boldanova, Tujana; Moes, Suzette; Adametz, David; Quagliata, Luca; Roth, Volker; Terracciano, Luigi; Heim, Markus H.; Jenoe, Paul; Hall, Michael N.

    2016-01-01

    Compensatory signaling pathways in tumors confer resistance to targeted therapy, but the pathways and their mechanisms of activation remain largely unknown. We describe a procedure for quantitative proteomics and phosphoproteomics on snap-frozen biopsies of hepatocellular carcinoma (HCC) and matched nontumor liver tissue. We applied this procedure to monitor signaling pathways in serial biopsies taken from an HCC patient before and during treatment with the multikinase inhibitor sorafenib. At diagnosis, the patient had an advanced HCC. At the time of the second biopsy, abdominal imaging revealed progressive disease despite sorafenib treatment. Sorafenib was confirmed to inhibit MAPK signaling in the tumor, as measured by reduced ribosomal protein S6 kinase phosphorylation. Hierarchical clustering and enrichment analysis revealed pathways broadly implicated in tumor progression and resistance, such as epithelial-to-mesenchymal transition and cell adhesion pathways. Thus, we describe a protocol for quantitative analysis of oncogenic pathways in HCC biopsies and obtained first insights into the effect of sorafenib in vivo. This protocol will allow elucidation of mechanisms of resistance and enable precision medicine. PMID:26787912

  16. Quantitative proteomics and phosphoproteomics on serial tumor biopsies from a sorafenib-treated HCC patient.

    PubMed

    Dazert, Eva; Colombi, Marco; Boldanova, Tujana; Moes, Suzette; Adametz, David; Quagliata, Luca; Roth, Volker; Terracciano, Luigi; Heim, Markus H; Jenoe, Paul; Hall, Michael N

    2016-02-01

    Compensatory signaling pathways in tumors confer resistance to targeted therapy, but the pathways and their mechanisms of activation remain largely unknown. We describe a procedure for quantitative proteomics and phosphoproteomics on snap-frozen biopsies of hepatocellular carcinoma (HCC) and matched nontumor liver tissue. We applied this procedure to monitor signaling pathways in serial biopsies taken from an HCC patient before and during treatment with the multikinase inhibitor sorafenib. At diagnosis, the patient had an advanced HCC. At the time of the second biopsy, abdominal imaging revealed progressive disease despite sorafenib treatment. Sorafenib was confirmed to inhibit MAPK signaling in the tumor, as measured by reduced ribosomal protein S6 kinase phosphorylation. Hierarchical clustering and enrichment analysis revealed pathways broadly implicated in tumor progression and resistance, such as epithelial-to-mesenchymal transition and cell adhesion pathways. Thus, we describe a protocol for quantitative analysis of oncogenic pathways in HCC biopsies and obtained first insights into the effect of sorafenib in vivo. This protocol will allow elucidation of mechanisms of resistance and enable precision medicine. PMID:26787912

  17. Quantitative Phosphoproteomics Analysis Reveals Broad Regulatory Role of Heparan Sulfate on Endothelial Signaling*

    PubMed Central

    Qiu, Hong; Jiang, Jun-Lin; Liu, Miao; Huang, Xin; Ding, Shi-Jian; Wang, Lianchun

    2013-01-01

    Heparan sulfate (HS) is a linear, abundant, highly sulfated polysaccharide that expresses in the vasculature. Recent genetic studies documented that HS critically modulates various endothelial cell functions. However, elucidation of the underlying molecular mechanism has been challenging because of the presence of a large number of HS-binding ligands found in the examined experimental conditions. In this report, we used quantitative phosphoproteomics to examine the global HS-dependent signaling by comparing wild type and HS-deficient endothelial cells that were cultured in a serum-containing medium. A total of 7222 phosphopeptides, corresponding to 1179 proteins, were identified. Functional correlation analysis identified 25 HS-dependent functional networks, and the top five are related to cell morphology, cellular assembly and organization, cellular function and maintenance, cell-to-cell communication, inflammatory response and disorder, cell growth and proliferation, cell movement, and cellular survival and death. This is consistent with cell function studies showing that HS deficiency altered endothelial cell growth and mobility. Mining for the underlying molecular mechanisms further revealed that HS modulates signaling pathways critically related to cell adhesion, migration, and coagulation, including ILK, integrin, actin cytoskeleton organization, tight junction and thrombin signaling. Intriguingly, this analysis unexpectedly determined that the top HS-dependent signaling is the IGF-1 signaling pathway, which has not been known to be modulated by HS. In-depth analysis of growth factor signaling identified 22 HS-dependent growth factor/cytokine/growth hormone signaling pathways, including those both previously known, such as HGF and VEGF, and those unknown, such as IGF-1, erythropoietin, angiopoietin/Tie, IL-17A and growth hormones. Twelve of the identified 22 growth factor/cytokine/growth hormone signaling pathways, including IGF-1 and angiopoietin

  18. Quantitative Proteomic and Phosphoproteomic Approaches for Deciphering the Signaling Pathway for Tension Wood Formation in Poplar.

    PubMed

    Mauriat, Mélanie; Leplé, Jean-Charles; Claverol, Stéphane; Bartholomé, Jérôme; Negroni, Luc; Richet, Nicolas; Lalanne, Céline; Bonneu, Marc; Coutand, Catherine; Plomion, Christophe

    2015-08-01

    Trees adjust their growth following forced changes in orientation to re-establish a vertical position. In angiosperms, this adjustment involves the differential regulation of vascular cambial activity between the lower (opposite wood) and upper (tension wood) sides of the leaning stem. We investigated the molecular mechanisms leading to the formation of differential wood types through a quantitative proteomic and phosphoproteomic analysis on poplar subjected to a gravitropic stimulus. We identified and quantified 675 phosphopeptides, corresponding to 468 phosphoproteins, and 3 763 nonphosphorylated peptides, corresponding to 1 155 proteins, in the differentiating xylem of straight-growing trees (control) and trees subjected to a gravitational stimulus during 8 weeks. About 1% of the peptides were specific to a wood type (straight, opposite, or tension wood). Proteins quantified in more than one type of wood were more numerous: a mixed linear model showed 389 phosphopeptides and 556 proteins to differ in abundance between tension wood and opposite wood. Twenty-one percent of the phosphoproteins identified here were described in their phosphorylated form for the first time. Our analyses revealed remarkable developmental molecular plasticity, with wood type-specific phosphorylation events, and highlighted the involvement of different proteins in the biosynthesis of cell wall components during the formation of the three types of wood.

  19. Quantitative Phosphoproteomic Profiling of Human Non-Small Cell Lung Cancer Tumors

    PubMed Central

    Schweppe, Devin K.; Rigas, James R.; Gerber, Scott A.

    2013-01-01

    Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide. Within the molecular scope of NCSLC, a complex landscape of dysregulated cellular signaling has emerged, defined largely by mutations in select mediators of signal transduction, including the epidermal growth factor receptor (EGFR) and anaplastic lymphoma (ALK) kinases. Consequently, these mutant kinases become constitutively activated and targets for chemotherapeutic intervention. Encouragingly, small molecule inhibitors of these pathways have shown promise in clinical trials or are approved for clinical use. However, many protein kinases are dysregulated in NSCLC without genetic mutations. To quantify differences in tumor cell signaling that are transparent to genomic methods, we established a super-SILAC internal standard derived from NSCLC cell lines grown in vitro and labeled with heavy lysine and arginine, and deployed them in a phosphoproteomics workflow. We identified 9019 and 8753 phosphorylation sites in two separate tumors. Relative quantification of phosphopeptide abundance between tumor samples allowed for the determination of specific hubs and pathways differing between each tumor. Sites downstream of Ras showed decreased inhibitory phosphorylation (Raf/Mek) and increased activating phosphorylation (Erk1/2) in one tumor versus another. In this way, we were able to quantitatively access oncogenic kinase signaling in primary human tumors. PMID:23911959

  20. Quantitative Proteomic and Phosphoproteomic Approaches for Deciphering the Signaling Pathway for Tension Wood Formation in Poplar.

    PubMed

    Mauriat, Mélanie; Leplé, Jean-Charles; Claverol, Stéphane; Bartholomé, Jérôme; Negroni, Luc; Richet, Nicolas; Lalanne, Céline; Bonneu, Marc; Coutand, Catherine; Plomion, Christophe

    2015-08-01

    Trees adjust their growth following forced changes in orientation to re-establish a vertical position. In angiosperms, this adjustment involves the differential regulation of vascular cambial activity between the lower (opposite wood) and upper (tension wood) sides of the leaning stem. We investigated the molecular mechanisms leading to the formation of differential wood types through a quantitative proteomic and phosphoproteomic analysis on poplar subjected to a gravitropic stimulus. We identified and quantified 675 phosphopeptides, corresponding to 468 phosphoproteins, and 3 763 nonphosphorylated peptides, corresponding to 1 155 proteins, in the differentiating xylem of straight-growing trees (control) and trees subjected to a gravitational stimulus during 8 weeks. About 1% of the peptides were specific to a wood type (straight, opposite, or tension wood). Proteins quantified in more than one type of wood were more numerous: a mixed linear model showed 389 phosphopeptides and 556 proteins to differ in abundance between tension wood and opposite wood. Twenty-one percent of the phosphoproteins identified here were described in their phosphorylated form for the first time. Our analyses revealed remarkable developmental molecular plasticity, with wood type-specific phosphorylation events, and highlighted the involvement of different proteins in the biosynthesis of cell wall components during the formation of the three types of wood. PMID:26112267

  1. Quantitative Phosphoproteomics Reveals the Role of Protein Arginine Phosphorylation in the Bacterial Stress Response*

    PubMed Central

    Schmidt, Andreas; Trentini, Débora Broch; Spiess, Silvia; Fuhrmann, Jakob; Ammerer, Gustav; Mechtler, Karl; Clausen, Tim

    2014-01-01

    Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response. PMID:24263382

  2. Quantitative Phosphoproteomics Identifies Filaggrin and other Targets of Ionizing Radiation in a Human Skin Model

    SciTech Connect

    Yang, Feng; Waters, Katrina M.; Webb-Robertson, Bobbie-Jo M.; Sowa, Marianne B.; Freiin von Neubeck, Claere H.; Aldrich, Joshua T.; Markillie, Lye Meng; Wirgau, Rachel M.; Gristenko, Marina A.; Zhao, Rui; Camp, David G.; Smith, Richard D.; Stenoien, David L.

    2012-04-17

    Our objective here was to perform a quantitative phosphoproteomic study on a reconstituted human skin tissue to identify low and high dose ionizing radiation dependent signaling in a complex 3-dimensional setting. Application of an isobaric labeling strategy using sham and 3 radiation doses (3, 10, 200 cGy) resulted in the identification of 1113 unique phosphopeptides. Statistical analyses identified 151 phosphopeptides showing significant changes in response to radiation and radiation dose. Proteins responsible for maintaining skin structural integrity including keratins and desmosomal proteins (desmoglein, desmoplakin, plakophilin 1 and 2,) had altered phosphorylation levels following exposure to both low and high doses of radiation. A phosphorylation site present in multiple copies in the linker regions of human profilaggrin underwent the largest fold change. Increased phosphorylation of these sites coincided with altered profilaggrin processing suggesting a role for linker phosphorylation in human profilaggrin regulation. These studies demonstrate that the reconstituted human skin system undergoes a coordinated response to ionizing radiation involving multiple layers of the stratified epithelium that serve to maintain skin barrier functions and minimize the damaging consequences of radiation exposure.

  3. Quantitative Phosphoproteome Profiling of Iron-Deficient Arabidopsis Roots1[C][W

    PubMed Central

    Lan, Ping; Li, Wenfeng; Wen, Tuan-Nan; Schmidt, Wolfgang

    2012-01-01

    Iron (Fe) is an essential mineral nutrient for plants, but often it is not available in sufficient quantities to sustain optimal growth. To gain insights into adaptive processes to low Fe availability at the posttranslational level, we conducted a quantitative analysis of Fe deficiency-induced changes in the phosphoproteome profile of Arabidopsis (Arabidopsis thaliana) roots. Isobaric tags for relative and absolute quantitation-labeled phosphopeptides were analyzed by liquid chromatography-tandem mass spectrometry on an LTQ-Orbitrap with collision-induced dissociation and high-energy collision dissociation capabilities. Using a combination of titanium dioxide and immobilized metal affinity chromatography to enrich phosphopeptides, we extracted 849 uniquely identified phosphopeptides corresponding to 425 proteins and identified several not previously described phosphorylation motifs. A subset of 45 phosphoproteins was defined as being significantly changed in abundance upon Fe deficiency. Kinase motifs in Fe-responsive proteins matched to protein kinase A/calcium calmodulin-dependent kinase II, casein kinase II, and proline-directed kinase, indicating a possible critical function of these kinase classes in Fe homeostasis. To validate our analysis, we conducted site-directed mutagenesis on IAA-CONJUGATE-RESISTANT4 (IAR4), a protein putatively functioning in auxin homeostasis. iar4 mutants showed compromised root hair formation and developed shorter primary roots. Changing serine-296 in IAR4 to alanine resulted in a phenotype intermediate between mutant and wild type, whereas acidic substitution to aspartate to mimic phosphorylation was either lethal or caused an extreme dwarf phenotype, supporting the critical importance of this residue in Fe homeostasis. Our analyses further disclose substantial changes in the abundance of phosphoproteins involved in primary carbohydrate metabolism upon Fe deficiency, complementing the picture derived from previous proteomic and

  4. Quantitative Phosphoproteomics Reveals Wee1 Kinase as a Therapeutic Target in a Model of Proneural Glioblastoma.

    PubMed

    Lescarbeau, Rebecca S; Lei, Liang; Bakken, Katrina K; Sims, Peter A; Sarkaria, Jann N; Canoll, Peter; White, Forest M

    2016-06-01

    Glioblastoma (GBM) is the most common malignant primary brain cancer. With a median survival of about a year, new approaches to treating this disease are necessary. To identify signaling molecules regulating GBM progression in a genetically engineered murine model of proneural GBM, we quantified phosphotyrosine-mediated signaling using mass spectrometry. Oncogenic signals, including phosphorylated ERK MAPK, PI3K, and PDGFR, were found to be increased in the murine tumors relative to brain. Phosphorylation of CDK1 pY15, associated with the G2 arrest checkpoint, was identified as the most differentially phosphorylated site, with a 14-fold increase in phosphorylation in the tumors. To assess the role of this checkpoint as a potential therapeutic target, syngeneic primary cell lines derived from these tumors were treated with MK-1775, an inhibitor of Wee1, the kinase responsible for CDK1 Y15 phosphorylation. MK-1775 treatment led to mitotic catastrophe, as defined by increased DNA damage and cell death by apoptosis. To assess the extensibility of targeting Wee1/CDK1 in GBM, patient-derived xenograft (PDX) cell lines were also treated with MK-1775. Although the response was more heterogeneous, on-target Wee1 inhibition led to decreased CDK1 Y15 phosphorylation and increased DNA damage and apoptosis in each line. These results were also validated in vivo, where single-agent MK-1775 demonstrated an antitumor effect on a flank PDX tumor model, increasing mouse survival by 1.74-fold. This study highlights the ability of unbiased quantitative phosphoproteomics to reveal therapeutic targets in tumor models, and the potential for Wee1 inhibition as a treatment approach in preclinical models of GBM. Mol Cancer Ther; 15(6); 1332-43. ©2016 AACR. PMID:27196784

  5. Quantitative phosphoproteomics of protein kinase SnRK1 regulated protein phosphorylation in Arabidopsis under submergence

    PubMed Central

    Cho, Hsing-Yi; Wen, Tuan-Nan; Wang, Ying-Tsui; Shih, Ming-Che

    2016-01-01

    SNF1 RELATED PROTEIN KINASE 1 (SnRK1) is proposed to be a central integrator of the plant stress and energy starvation signalling pathways. We observed that the Arabidopsis SnRK1.1 dominant negative mutant (SnRK1.1 K48M) had lower tolerance to submergence than the wild type, suggesting that SnRK1.1-dependent phosphorylation of target proteins is important in signalling pathways triggered by submergence. We conducted quantitative phosphoproteomics and found that the phosphorylation levels of 57 proteins increased and the levels of 27 proteins decreased in Col-0 within 0.5–3h of submergence. Among the 57 proteins with increased phosphorylation in Col-0, 38 did not show increased phosphorylation levels in SnRK1.1 K48M under submergence. These proteins are involved mainly in sugar and protein synthesis. In particular, the phosphorylation of MPK6, which is involved in regulating ROS responses under abiotic stresses, was disrupted in the SnRK1.1 K48M mutant. In addition, PTP1, a negative regulator of MPK6 activity that directly dephosphorylates MPK6, was also regulated by SnRK1.1. We also showed that energy conservation was disrupted in SnRK1.1 K48M, mpk6, and PTP1 S7AS8A under submergence. These results reveal insights into the function of SnRK1 and the downstream signalling factors related to submergence. PMID:27029354

  6. Quantitative Phosphoproteomics Reveals Crosstalk Between Phosphorylation and O-GlcNAc in the DNA Damage Response Pathway

    PubMed Central

    Zhong, Jun; Martinez, Marissa; Sengupta, Srona; Lee, Albert; Wu, Xinyan; Chaerkady, Raghothama; Chatterjee, Aditi; O’Meally, Robert N.; Cole, Robert N.; Pandey, Akhilesh; Zachara, Natasha E.

    2015-01-01

    The modification of intracellular proteins by monosaccharides of O-linked β-N-acetylglucosamine (O-GlcNAc) is an essential and dynamic post-translational modification of metazoans. The addition and removal of O-GlcNAc is catalyzed by the O-GlcNAc transferase (OGT) and O-GlcNAcase, respectively. One mechanism by which O-GlcNAc is thought to mediate proteins is by regulating phosphorylation. To provide insight into the pathways regulated by O-GlcNAc, we have utilized stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative proteomics to carry out comparisons of site-specific phosphorylation in OGT wild-type (WT) and Null cells. Quantitation of the phosphoproteome demonstrated that out of 5,529 phosphoserine, phosphothreonine and phosphotyrosine sites, 232 phosphosites were upregulated and 133 downregulated in the absence of O-GlcNAc. Collectively, these data suggest that deletion of OGT has a profound effect on the phosphorylation of cell cycle and DNA damage response proteins. Key events were confirmed by biochemical analyses and demonstrate a increase in the activating autophosphorylation event on ATM (Ser1987) and on ATM’s downstream targets p53, H2AX and Chk2. Together, these data support widespread changes in the phosphoproteome upon removal of O-GlcNAc, suggesting that O-GlcNAc regulates processes such as the cell cycle, genomic stability, and lysosomal biogenesis. PMID:25263469

  7. Quantitative Label-Free Phosphoproteomics Reveals Differentially Regulated Protein Phosphorylation Involved in West Nile Virus-Induced Host Inflammatory Response.

    PubMed

    Zhang, Hao; Sun, Jun; Ye, Jing; Ashraf, Usama; Chen, Zheng; Zhu, Bibo; He, Wen; Xu, Qiuping; Wei, Yanming; Chen, Huanchun; Fu, Zhen F; Liu, Rong; Cao, Shengbo

    2015-12-01

    West Nile virus (WNV) can cause neuro-invasive and febrile illness that may be fatal to humans. The production of inflammatory cytokines is key to mediating WNV-induced immunopathology in the central nervous system. Elucidating the host factors utilized by WNV for productive infection would provide valuable insights into the evasion strategies used by this virus. Although attempts have been made to determine these host factors, proteomic data depicting WNV-host protein interactions are limited. We applied liquid chromatography-tandem mass spectrometry for label-free, quantitative phosphoproteomics to systematically investigate the global phosphorylation events induced by WNV infection. Quantifiable changes to 1,657 phosphoproteins were found; of these, 626 were significantly upregulated and 227 were downregulated at 12 h postinfection. The phosphoproteomic data were subjected to gene ontology enrichment analysis, which returned the inflammation-related spliceosome, ErbB, mitogen-activated protein kinase, nuclear factor kappa B, and mechanistic target of rapamycin signaling pathways. We used short interfering RNAs to decrease the levels of glycogen synthase kinase-3 beta, bifunctional polynucleotide phosphatase/kinase, and retinoblastoma 1 and found that the activity of nuclear factor kappa B (p65) is significantly decreased in WNV-infected U251 cells, which in turn led to markedly reduced inflammatory cytokine production. Our results provide a better understanding of the host response to WNV infection and highlight multiple targets for the development of antiviral and anti-inflammatory therapies.

  8. Multidimensional electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) for quantitative analysis of the proteome and phosphoproteome in clinical and biomedical research.

    PubMed

    Loroch, Stefan; Schommartz, Tim; Brune, Wolfram; Zahedi, René Peiman; Sickmann, Albert

    2015-05-01

    Quantitative proteomics and phosphoproteomics have become key disciplines in understanding cellular processes. Fundamental research can be done using cell culture providing researchers with virtually infinite sample amounts. In contrast, clinical, pre-clinical and biomedical research is often restricted to minute sample amounts and requires an efficient analysis with only micrograms of protein. To address this issue, we generated a highly sensitive workflow for combined LC-MS-based quantitative proteomics and phosphoproteomics by refining an ERLIC-based 2D phosphoproteomics workflow into an ERLIC-based 3D workflow covering the global proteome as well. The resulting 3D strategy was successfully used for an in-depth quantitative analysis of both, the proteome and the phosphoproteome of murine cytomegalovirus-infected mouse fibroblasts, a model system for host cell manipulation by a virus. In a 2-plex SILAC experiment with 150 μg of a tryptic digest per condition, the 3D strategy enabled the quantification of ~75% more proteins and even ~134% more peptides compared to the 2D strategy. Additionally, we could quantify ~50% more phosphoproteins by non-phosphorylated peptides, concurrently yielding insights into changes on the levels of protein expression and phosphorylation. Beside its sensitivity, our novel three-dimensional ERLIC-strategy has the potential for semi-automated sample processing rendering it a suitable future perspective for clinical, pre-clinical and biomedical research.

  9. Multidimensional electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) for quantitative analysis of the proteome and phosphoproteome in clinical and biomedical research.

    PubMed

    Loroch, Stefan; Schommartz, Tim; Brune, Wolfram; Zahedi, René Peiman; Sickmann, Albert

    2015-05-01

    Quantitative proteomics and phosphoproteomics have become key disciplines in understanding cellular processes. Fundamental research can be done using cell culture providing researchers with virtually infinite sample amounts. In contrast, clinical, pre-clinical and biomedical research is often restricted to minute sample amounts and requires an efficient analysis with only micrograms of protein. To address this issue, we generated a highly sensitive workflow for combined LC-MS-based quantitative proteomics and phosphoproteomics by refining an ERLIC-based 2D phosphoproteomics workflow into an ERLIC-based 3D workflow covering the global proteome as well. The resulting 3D strategy was successfully used for an in-depth quantitative analysis of both, the proteome and the phosphoproteome of murine cytomegalovirus-infected mouse fibroblasts, a model system for host cell manipulation by a virus. In a 2-plex SILAC experiment with 150 μg of a tryptic digest per condition, the 3D strategy enabled the quantification of ~75% more proteins and even ~134% more peptides compared to the 2D strategy. Additionally, we could quantify ~50% more phosphoproteins by non-phosphorylated peptides, concurrently yielding insights into changes on the levels of protein expression and phosphorylation. Beside its sensitivity, our novel three-dimensional ERLIC-strategy has the potential for semi-automated sample processing rendering it a suitable future perspective for clinical, pre-clinical and biomedical research. PMID:25619855

  10. Quantitative Phosphoproteomic Analysis of Soybean Root Hairs Inoculated with Bradyrhizobium japonicum

    SciTech Connect

    Nguyen, Tran H.; Brechenmacher, Laurent; Aldrich, Joshua T.; Clauss, Therese RW; Gritsenko, Marina A.; Hixson, Kim K.; Libault, Marc; Tanaka, Kiwamu; Yang, Feng; Yao, Qiuming; Pasa-Tolic, Ljiljana; Xu, Dong; Nguyen, Henry T.; Stacey, Gary

    2012-11-11

    Root hairs are single hair-forming cells on roots that function to increase root surface area, enhancing water and nutrient uptake. In leguminous plants, root hairs also play a critical role as the site of infection by symbiotic nitrogen fixing rhizobia, leading to the formation of a novel organ, the nodule. The initial steps in the rhizobia-root hair infection process are known to involve specific receptor kinases and subsequent kinase cascades. Here, we characterize the phosphoproteome of the root hairs and the corresponding stripped roots (i.e., roots from which root hairs were removed) during rhizobial colonization and infection to gain insight into the molecular mechanism of root hair cell biology. We chose soybean (Glycine max L.), one of the most important crop plants in the legume family, for this study because of its larger root size, which permits isolation of sufficient root hair material for phosphoproteomic analysis. Phosphopeptides derived from root hairs and stripped roots, mock inoculated or inoculated with the soybean-specific rhizobium Bradyrhizobium japonicum, were labeled with the isobaric tag 8-plex ITRAQ, enriched using Ni-NTA magnetic beads and subjected to nRPLC-MS/MS analysis using HCD and decision tree guided CID/ETD strategy. A total of 1,625 unique phosphopeptides, spanning 1,659 non-redundant phosphorylation sites, were detected from 1,126 soybean phosphoproteins. Among them, 273 phosphopeptides corresponding to 240 phosphoproteins were found to be significantly regulated (>1.5 fold abundance change) in response to inoculation with B. japonicum. The data reveal unique features of the soybean root hair phosphoproteome, including root hair and stripped root-specific phosphorylation suggesting a complex network of kinase-substrate and phosphatase-substrate interactions in response to rhizobial inoculation.

  11. Identification of Putative Mek1 Substrates during Meiosis in Saccharomyces cerevisiae Using Quantitative Phosphoproteomics.

    PubMed

    Suhandynata, Raymond T; Wan, Lihong; Zhou, Huilin; Hollingsworth, Nancy M

    2016-01-01

    Meiotic recombination plays a key role in sexual reproduction as it generates crossovers that, in combination with sister chromatid cohesion, physically connect homologous chromosomes, thereby promoting their proper segregation at the first meiotic division. Meiotic recombination is initiated by programmed double strand breaks (DSBs) catalyzed by the evolutionarily conserved, topoisomerase-like protein Spo11. Repair of these DSBs is highly regulated to create crossovers between homologs that are distributed throughout the genome. This repair requires the presence of the mitotic recombinase, Rad51, as well as the strand exchange activity of the meiosis-specific recombinase, Dmc1. A key regulator of meiotic DSB repair in Saccharomyces cerevisiae is the meiosis-specific kinase Mek1, which promotes interhomolog strand invasion and is required for the meiotic recombination checkpoint and the crossover/noncrossover decision. Understanding how Mek1 regulates meiotic recombination requires the identification of its substrates. Towards that end, an unbiased phosphoproteomic approach utilizing Stable Isotope Labeling by Amino Acids in Cells (SILAC) was utilized to generate a list of potential Mek1 substrates, as well as proteins containing consensus phosphorylation sites for cyclin-dependent kinase, the checkpoint kinases, Mec1/Tel1, and the polo-like kinase, Cdc5. These experiments represent the first global phosphoproteomic dataset for proteins in meiotic budding yeast.

  12. Quantitative Phosphoproteomics Reveals Signaling Mechanisms Associated with Rapid Cold Hardening in a Chill-Tolerant Fly.

    PubMed

    Teets, Nicholas M; Denlinger, David L

    2016-08-01

    Rapid cold hardening (RCH) is a physiological adaptation in which brief chilling (minutes to hours) significantly enhances the cold tolerance of insects. RCH allows insects to cope with sudden cold snaps and diurnal variation in temperature, but the mechanistic basis of this rapid stress response is poorly understood. Here, we used phosphoproteomics to identify phosphorylation-mediated signaling events that are regulated by chilling that induces RCH. Phosphoproteomic changes were measured in both brain and fat bodies, two tissues that are essential for sensing cold and coordinating RCH at the organismal level. Tissues were chilled ex vivo, and changes in phosphoprotein abundance were measured using 2D electrophoresis coupled with Pro-Q diamond labeling of phosphoproteins followed by protein identification via LC-MS/MS. In both tissues, we observed an abundance of protein phosphorylation events in response to chilling. Some of the proteins regulated by RCH-inducing chilling include proteins involved in cytoskeletal reorganization, heat shock proteins, and proteins involved in the degradation of damaged cellular components via the proteasome and autophagosome. Our results suggest that phosphorylation-mediated signaling cascades are major drivers of RCH and enhance our mechanistic understanding of this complex phenotype.

  13. Quantitative analysis of changes in the phosphoproteome of maize induced by the plant hormone salicylic acid

    PubMed Central

    Wu, Liuji; Hu, Xiuli; Wang, Shunxi; Tian, Lei; Pang, Yanjie; Han, Zanping; Wu, Liancheng; Chen, Yanhui

    2015-01-01

    Phytohormone salicylic acid (SA) plays an important role in regulating various physiological and biochemical processes. Our previous study identified several protein kinases responsive to SA, suggesting that phosphorylation events play an important role in the plant response to SA. In this study, we characterized the phosphoproteome of maize in response to SA using isotope tags for relative and absolute quantification (iTRAQ) technology and TiO2 enrichment method. Based on LC-MS/MS analysis, we found a total of 858 phosphoproteins among 1495 phosphopeptides. Among them, 291 phosphopeptides corresponding to 244 phosphoproteins were found to be significantly changed after SA treatment. The phosphoproteins identified are involved in a wide range of biological processes, which indicate that the response to SA encompasses a reformatting of major cellular processes. Furthermore, some of the phosphoproteins which were not previously known to be involved with SA were found to have significantly changed phosphorylation levels. Many of these changes are phosphorylation decreases, indicating that other currently unknown SA signaling pathways that result in decreased phosphorylation of downstream targets must be involved. Our study represents the first attempt at global phosphoproteome profiling in response to SA, and provides a better understanding of the molecular mechanisms regulated by SA. PMID:26659305

  14. Identification of Putative Mek1 Substrates during Meiosis in Saccharomyces cerevisiae Using Quantitative Phosphoproteomics

    PubMed Central

    Suhandynata, Raymond T.; Wan, Lihong; Zhou, Huilin; Hollingsworth, Nancy M.

    2016-01-01

    Meiotic recombination plays a key role in sexual reproduction as it generates crossovers that, in combination with sister chromatid cohesion, physically connect homologous chromosomes, thereby promoting their proper segregation at the first meiotic division. Meiotic recombination is initiated by programmed double strand breaks (DSBs) catalyzed by the evolutionarily conserved, topoisomerase-like protein Spo11. Repair of these DSBs is highly regulated to create crossovers between homologs that are distributed throughout the genome. This repair requires the presence of the mitotic recombinase, Rad51, as well as the strand exchange activity of the meiosis-specific recombinase, Dmc1. A key regulator of meiotic DSB repair in Saccharomyces cerevisiae is the meiosis-specific kinase Mek1, which promotes interhomolog strand invasion and is required for the meiotic recombination checkpoint and the crossover/noncrossover decision. Understanding how Mek1 regulates meiotic recombination requires the identification of its substrates. Towards that end, an unbiased phosphoproteomic approach utilizing Stable Isotope Labeling by Amino Acids in Cells (SILAC) was utilized to generate a list of potential Mek1 substrates, as well as proteins containing consensus phosphorylation sites for cyclin-dependent kinase, the checkpoint kinases, Mec1/Tel1, and the polo-like kinase, Cdc5. These experiments represent the first global phosphoproteomic dataset for proteins in meiotic budding yeast. PMID:27214570

  15. Quantitative Phosphoproteomics Reveals Signaling Mechanisms Associated with Rapid Cold Hardening in a Chill-Tolerant Fly.

    PubMed

    Teets, Nicholas M; Denlinger, David L

    2016-08-01

    Rapid cold hardening (RCH) is a physiological adaptation in which brief chilling (minutes to hours) significantly enhances the cold tolerance of insects. RCH allows insects to cope with sudden cold snaps and diurnal variation in temperature, but the mechanistic basis of this rapid stress response is poorly understood. Here, we used phosphoproteomics to identify phosphorylation-mediated signaling events that are regulated by chilling that induces RCH. Phosphoproteomic changes were measured in both brain and fat bodies, two tissues that are essential for sensing cold and coordinating RCH at the organismal level. Tissues were chilled ex vivo, and changes in phosphoprotein abundance were measured using 2D electrophoresis coupled with Pro-Q diamond labeling of phosphoproteins followed by protein identification via LC-MS/MS. In both tissues, we observed an abundance of protein phosphorylation events in response to chilling. Some of the proteins regulated by RCH-inducing chilling include proteins involved in cytoskeletal reorganization, heat shock proteins, and proteins involved in the degradation of damaged cellular components via the proteasome and autophagosome. Our results suggest that phosphorylation-mediated signaling cascades are major drivers of RCH and enhance our mechanistic understanding of this complex phenotype. PMID:27362561

  16. Quantitative phosphoproteomic analysis of the PI3K-regulated signaling network.

    PubMed

    Gnad, Florian; Wallin, Jeffrey; Edgar, Kyle; Doll, Sophia; Arnott, David; Robillard, Liliane; Kirkpatrick, Donald S; Stokes, Matthew P; Vijapurkar, Ulka; Hatzivassiliou, Georgia; Friedman, Lori S; Belvin, Marcia

    2016-07-01

    The PI3K pathway is commonly activated in cancer. Only a few studies have attempted to explore the spectrum of phosphorylation signaling downstream of the PI3K cascade. Such insight, however, is imperative to understand the mechanisms responsible for oncogenic phenotypes. By applying MS-based phosphoproteomics, we mapped 2509 phosphorylation sites on 1096 proteins, and quantified their responses to activation or inhibition of PIK3CA using isogenic knock-in derivatives and a series of targeted inhibitors. We uncovered phosphorylation changes in a wide variety of proteins involved in cell growth and proliferation, many of which have not been previously associated with PI3K signaling. A significant update of the posttranslational modification database PHOSIDA (http://www.phosida.com) allows efficient use of the data. All MS data have been deposited in the ProteomeXchange with identifier PXD003899 (http://proteomecentral.proteomexchange.org/dataset/PXD003899). PMID:27282143

  17. Quantitative phosphoproteomics in nuclei of vasopressin-sensitive renal collecting duct cells

    PubMed Central

    Bolger, Steven J.; Hurtado, Patricia A. Gonzales; Hoffert, Jason D.; Saeed, Fahad; Pisitkun, Trairak

    2012-01-01

    Vasopressin regulates transport across the collecting duct epithelium in part via effects on gene transcription. Transcriptional regulation occurs partially via changes in phosphorylation of transcription factors, transcriptional coactivators, and protein kinases in the nucleus. To test whether vasopressin alters the nuclear phosphoproteome of vasopressin-sensitive cultured mouse mpkCCD cells, we used stable isotope labeling and mass spectrometry to quantify thousands of phosphorylation sites in nuclear extracts and nuclear pellet fractions. Measurements were made in the presence and absence of the vasopressin analog dDAVP. Of the 1,251 sites quantified, 39 changed significantly in response to dDAVP. Network analysis of the regulated proteins revealed two major clusters (“cell-cell adhesion” and “transcriptional regulation”) that were connected to known elements of the vasopressin signaling pathway. The hub proteins for these two clusters were the transcriptional coactivator β-catenin and the transcription factor c-Jun. Phosphorylation of β-catenin at Ser552 was increased by dDAVP [log2(dDAVP/vehicle) = 1.79], and phosphorylation of c-Jun at Ser73 was decreased [log2(dDAVP/vehicle) = −0.53]. The β-catenin site is known to be targeted by either protein kinase A or Akt, both of which are activated in response to vasopressin. The c-Jun site is a canonical target for the MAP kinase Jnk2, which is downregulated in response to vasopressin in the collecting duct. The data support the idea that vasopressin-mediated control of transcription in collecting duct cells involves selective changes in the nuclear phosphoproteome. All data are available to users at http://helixweb.nih.gov/ESBL/Database/mNPPD/. PMID:22992673

  18. Quantitative comparison of the fasted and re-fed mouse liver phosphoproteomes using lower pH reductive dimethylation.

    PubMed

    Wilson-Grady, Joshua T; Haas, Wilhelm; Gygi, Steven P

    2013-06-15

    Phosphorylation is a common but crucial protein posttranslational modification occurring in virtually all known species. A successful technique for identifying phosphorylation sites is via liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition to identification, the introduction of stable isotopes allows for LC-MS based quantification of thousands of phosphorylation sites. Historically, stable isotope labeling by amino acids in cell culture (SILAC) has been the preferred method for introducing stable isotopes for quantification. SILAC is not well suited, however, for quantitative proteomics in larger animals. The introduction of stable isotope instead by reductive dimethylation is an alternative for performing quantitative proteomics in animal tissues. Here we present an improved reductive dimethylation protocol and demonstrate the application of this method in the analysis of the fasted vs. re-fed mouse liver phosphoproteome. In our analysis, greater than 8500 sites were identified from ∼2700 phosphoproteins. Nearly 7400 phosphorylation events from ∼2300 phosphoproteins were reliably quantified. Using a 2-fold change as a cutoff, 390 phosphorylation sites were found to change between fasted and re-fed mice, many of which may have interesting biological interpretations.

  19. Quantitative phosphoproteomic analyses of the inferior parietal lobule from three different pathological stages of Alzheimer's disease.

    PubMed

    Triplett, Judy C; Swomley, Aaron M; Cai, Jian; Klein, Jon B; Butterfield, D Allan

    2015-01-01

    Alzheimer's disease (AD), the most common age-related neurodegenerative disorder, is clinically characterized by progressive neuronal loss resulting in loss of memory and dementia. AD is histopathologically characterized by the extensive distribution of senile plaques and neurofibrillary tangles, and synapse loss. Amnestic mild cognitive impairment (MCI) is generally accepted to be an early stage of AD. MCI subjects have pathology and symptoms that fall on the scale intermediately between 'normal' cognition with little or no pathology and AD. A rare number of individuals, who exhibit normal cognition on psychometric tests but whose brains show widespread postmortem AD pathology, are classified as 'asymptomatic' or 'preclinical' AD (PCAD). In this study, we evaluated changes in protein phosphorylation states in the inferior parietal lobule of subjects with AD, MCI, PCAD, and control brain using a 2-D PAGE proteomics approach in conjunction with Pro-Q Diamond phosphoprotein staining. Statistically significant changes in phosphorylation levels were found in 19 proteins involved in energy metabolism, neuronal plasticity, signal transduction, and oxidative stress response. Changes in the disease state phosphoproteome may provide insights into underlying mechanisms for the preservation of memory with expansive AD pathology in PCAD and the progressive memory loss in amnestic MCI that escalates to the dementia and the characteristic pathology of AD brain.

  20. Quantitative phosphoproteomics of murine Fmr1-KO cell lines provides new insights into FMRP-dependent signal transduction mechanisms.

    PubMed

    Matic, Katarina; Eninger, Timo; Bardoni, Barbara; Davidovic, Laetitia; Macek, Boris

    2014-10-01

    Fragile X mental retardation protein (FMRP) is an RNA-binding protein that has a major effect on neuronal protein synthesis. Transcriptional silencing of the FMR1 gene leads to loss of FMRP and development of Fragile X syndrome (FXS), the most common known hereditary cause of intellectual impairment and autism. Here we utilize SILAC-based quantitative phosphoproteomics to analyze murine FMR1(-) and FMR1(+) fibroblastic cell lines derived from FMR1-KO embryos to identify proteins and phosphorylation sites dysregulated as a consequence of FMRP loss. We quantify FMRP-related changes in the levels of 5,023 proteins and 6,133 phosphorylation events and map them onto major signal transduction pathways. Our study confirms global downregulation of the MAPK/ERK pathway and decrease in phosphorylation level of ERK1/2 in the absence of FMRP, which is connected to attenuation of long-term potentiation. We detect differential expression of several key proteins from the p53 pathway, pointing to the involvement of p53 signaling in dysregulated cell cycle control in FXS. Finally, we detect differential expression and phosphorylation of proteins involved in pre-mRNA processing and nuclear transport, as well as Wnt and calcium signaling, such as PLC, PKC, NFAT, and cPLA2. We postulate that calcium homeostasis is likely affected in molecular pathogenesis of FXS. PMID:25168779

  1. Effects of MEK inhibitors GSK1120212 and PD0325901 in vivo using 10-plex quantitative proteomics and phosphoproteomics

    PubMed Central

    Paulo, Joao A.; McAllister, Fiona E.; Everley, Robert A.; Beausoleil, Sean A.; Banks, Alexander S.; Gygi, Steven P.

    2015-01-01

    Multiplexed isobaric tag-based quantitative proteomics and phosphoproteomics strategies can comprehensively analyze drug treatments effects on biological systems. Given the role of MEK signaling in cancer and MAPK-dependent diseases, we sought to determine if this pathway could be inhibited safely by examining the downstream molecular consequences. We used a series of TMT10-plex experiments to analyze the effect of two MEK inhibitors (GSK1120212 and PD0325901) on three tissues (kidney, liver, and pancreas) from nine mice. We quantified ~6000 proteins in each tissue, but significant protein level alterations were minimal with inhibitor treatment. Of particular interest was kidney tissue, as edema is an adverse effect of these inhibitors. From kidney tissue, we enriched phosphopeptides using titanium dioxide (TiO2) and quantified 10,562 phosphorylation events. Further analysis by phosphotyrosine (pY) peptide immunoprecipitation quantified an additional 592 phosphorylation events. Phosphorylation motif analysis revealed that the inhibitors decreased phosphorylation levels of PxSP and SP sites, consistent with ERK inhibition. The MEK inhibitors had the greatest decrease on the phosphorylation of two proteins, Barttin and Slc12a3, which have roles in ion transport and fluid balance. Further studies will provide insight into the effect of these MEK inhibitors with respect to edema and other adverse events in mouse models and human patients. PMID:25195567

  2. Quantitative phosphoproteomics revealed interplay between Syk and Lyn in the resistance to nilotinib in chronic myeloid leukemia cells.

    PubMed

    Gioia, Romain; Leroy, Cédric; Drullion, Claire; Lagarde, Valérie; Etienne, Gabriel; Dulucq, Stéphanie; Lippert, Eric; Roche, Serge; Mahon, François-Xavier; Pasquet, Jean-Max

    2011-08-25

    In this study, we have addressed how Lyn kinase signaling mediates nilotinib-resistance by quantitative phospho-proteomics using Stable Isotope Labeling with Amino acid in Cell culture. We have found an increased tyrosine phosphorylation of 2 additional tyrosine kinases in nilotinib-resistant cells: the spleen tyrosine kinase Syk and the UFO family receptor tyrosine kinase Axl. This increased tyrosine phosphorylation involved an interaction of these tyrosine kinases with Lyn. Inhibition of Syk by the inhibitors R406 or BAY 61-3606 or by RNA interference restored the capacity of nilotinib to inhibit cell proliferation. Conversely, coexpression of Lyn and Syk were required to fully induce resistance to nilotinib in drug-sensitive cells. Surprisingly, the knockdown of Syk also strongly decreased tyrosine phosphorylation of Lyn and Axl, thus uncovering interplay between Syk and Lyn. We have shown the involvement of the adaptor protein CDCP-1 in resistance to nilotinib. Interestingly, the expression of Axl and CDCP1 were found increased both in a nilotinib-resistant cell line and in nilotinib-resistant CML patients. We conclude that an oncogenic signaling mediated by Lyn and Syk can bypass the need of Bcr-Abl in CML cells. Thus, targeting these kinases may be of therapeutic value to override imatinib or nilotinib resistance in CML. PMID:21730355

  3. Quantitative phosphoproteomics of murine Fmr1-KO cell lines provides new insights into FMRP-dependent signal transduction mechanisms.

    PubMed

    Matic, Katarina; Eninger, Timo; Bardoni, Barbara; Davidovic, Laetitia; Macek, Boris

    2014-10-01

    Fragile X mental retardation protein (FMRP) is an RNA-binding protein that has a major effect on neuronal protein synthesis. Transcriptional silencing of the FMR1 gene leads to loss of FMRP and development of Fragile X syndrome (FXS), the most common known hereditary cause of intellectual impairment and autism. Here we utilize SILAC-based quantitative phosphoproteomics to analyze murine FMR1(-) and FMR1(+) fibroblastic cell lines derived from FMR1-KO embryos to identify proteins and phosphorylation sites dysregulated as a consequence of FMRP loss. We quantify FMRP-related changes in the levels of 5,023 proteins and 6,133 phosphorylation events and map them onto major signal transduction pathways. Our study confirms global downregulation of the MAPK/ERK pathway and decrease in phosphorylation level of ERK1/2 in the absence of FMRP, which is connected to attenuation of long-term potentiation. We detect differential expression of several key proteins from the p53 pathway, pointing to the involvement of p53 signaling in dysregulated cell cycle control in FXS. Finally, we detect differential expression and phosphorylation of proteins involved in pre-mRNA processing and nuclear transport, as well as Wnt and calcium signaling, such as PLC, PKC, NFAT, and cPLA2. We postulate that calcium homeostasis is likely affected in molecular pathogenesis of FXS.

  4. Effects of MEK inhibitors GSK1120212 and PD0325901 in vivo using 10-plex quantitative proteomics and phosphoproteomics.

    PubMed

    Paulo, Joao A; McAllister, Fiona E; Everley, Robert A; Beausoleil, Sean A; Banks, Alexander S; Gygi, Steven P

    2015-01-01

    Multiplexed isobaric tag based quantitative proteomics and phosphoproteomics strategies can comprehensively analyze drug treatments effects on biological systems. Given the role of mitogen-activated protein/extracellular signal-regulated kinase (MEK) signaling in cancer and mitogen-activated protein kinase (MAPK)-dependent diseases, we sought to determine if this pathway could be inhibited safely by examining the downstream molecular consequences. We used a series of tandem mass tag 10-plex experiments to analyze the effect of two MEK inhibitors (GSK1120212 and PD0325901) on three tissues (kidney, liver, and pancreas) from nine mice. We quantified ∼ 6000 proteins in each tissue, but significant protein-level alterations were minimal with inhibitor treatment. Of particular interest was kidney tissue, as edema is an adverse effect of these inhibitors. From kidney tissue, we enriched phosphopeptides using titanium dioxide (TiO2 ) and quantified 10 562 phosphorylation events. Further analysis by phosphotyrosine peptide immunoprecipitation quantified an additional 592 phosphorylation events. Phosphorylation motif analysis revealed that the inhibitors decreased phosphorylation levels of proline-x-serine-proline (PxSP) and serine-proline (SP) sites, consistent with extracellular-signal-regulated kinase (ERK) inhibition. The MEK inhibitors had the greatest decrease on the phosphorylation of two proteins, Barttin and Slc12a3, which have roles in ion transport and fluid balance. Further studies will provide insight into the effect of these MEK inhibitors with respect to edema and other adverse events in mouse models and human patients.

  5. KinasePA: Phosphoproteomics data annotation using hypothesis driven kinase perturbation analysis

    PubMed Central

    Yang, Pengyi; Patrick, Ellis; Humphrey, Sean J.; Ghazanfar, Shila; James, David E.; Jothi, Raja; Yang, Jean Yee Hwa

    2016-01-01

    Mass spectrometry (MS)-based quantitative phosphoproteomics has become a key approach for proteome-wide profiling of phosphorylation in tissues and cells. Traditional experimental design often compares a single treatment with a control, whereas increasingly more experiments are designed to compare multiple treatments with respect to a control. To this end, the development of bioinformatic tools that can integrate multiple treatments and visualise kinases and substrates under combinatorial perturbations is vital for dissecting concordant and/or independent effects of each treatment. Here, we propose a hypothesis driven kinase perturbation analysis (KinasePA) to annotate and visualise kinases and their substrates that are perturbed by various combinatorial effects of treatments in phosphoproteomics experiments. We demonstrate the utility of KinasePA through its application to two large-scale phosphoproteomics datasets and show its effectiveness in dissecting kinases and substrates within signalling pathways driven by unique combinations of cellular stimuli and inhibitors. We implemented and incorporated KinasePA as part of the “directPA” R package available from the comprehensive R archive network (CRAN). Furthermore, KinasePA also has an interactive web interface that can be readily applied to annotate user provided phosphoproteomics data (http://kinasepa.pengyiyang.org). PMID:27145998

  6. KinasePA: Phosphoproteomics data annotation using hypothesis driven kinase perturbation analysis.

    PubMed

    Yang, Pengyi; Patrick, Ellis; Humphrey, Sean J; Ghazanfar, Shila; James, David E; Jothi, Raja; Yang, Jean Yee Hwa

    2016-07-01

    Mass spectrometry (MS)-based quantitative phosphoproteomics has become a key approach for proteome-wide profiling of phosphorylation in tissues and cells. Traditional experimental design often compares a single treatment with a control, whereas increasingly more experiments are designed to compare multiple treatments with respect to a control. To this end, the development of bioinformatic tools that can integrate multiple treatments and visualise kinases and substrates under combinatorial perturbations is vital for dissecting concordant and/or independent effects of each treatment. Here, we propose a hypothesis driven kinase perturbation analysis (KinasePA) to annotate and visualise kinases and their substrates that are perturbed by various combinatorial effects of treatments in phosphoproteomics experiments. We demonstrate the utility of KinasePA through its application to two large-scale phosphoproteomics datasets and show its effectiveness in dissecting kinases and substrates within signalling pathways driven by unique combinations of cellular stimuli and inhibitors. We implemented and incorporated KinasePA as part of the "directPA" R package available from the comprehensive R archive network (CRAN). Furthermore, KinasePA also has an interactive web interface that can be readily applied to annotate user provided phosphoproteomics data (http://kinasepa.pengyiyang.org).

  7. Quantitative Circadian Phosphoproteomic Analysis of Arabidopsis Reveals Extensive Clock Control of Key Components in Physiological, Metabolic, and Signaling Pathways.

    PubMed

    Choudhary, Mani Kant; Nomura, Yuko; Wang, Lei; Nakagami, Hirofumi; Somers, David E

    2015-08-01

    The circadian clock provides adaptive advantages to an organism, resulting in increased fitness and survival. The phosphorylation events that regulate circadian-dependent signaling and the processes which post-translationally respond to clock-gated signals are largely unknown. To better elucidate post-translational events tied to the circadian system we carried out a survey of circadian-regulated protein phosphorylation events in Arabidopsis seedlings. A large-scale mass spectrometry-based quantitative phosphoproteomics approach employing TiO2-based phosphopeptide enrichment techniques identified and quantified 1586 phosphopeptides on 1080 protein groups. A total of 102 phosphopeptides displayed significant changes in abundance, enabling the identification of specific patterns of response to circadian rhythms. Our approach was sensitive enough to quantitate oscillations in the phosphorylation of low abundance clock proteins (early flowering4; ELF4 and pseudoresponse regulator3; PRR3) as well as other transcription factors and kinases. During constant light, extensive cyclic changes in phosphorylation status occurred in critical regulators, implicating direct or indirect regulation by the circadian system. These included proteins influencing transcriptional regulation, translation, metabolism, stress and phytohormones-mediated responses. We validated our analysis using the elf4-211 allele, in which an S45L transition removes the phosphorylation herein identified. We show that removal of this phosphorylatable site diminishes interaction with early flowering3 (ELF3), a key partner in a tripartite evening complex required for circadian cycling. elf4-211 lengthens period, which increases with increasing temperature, relative to the wild type, resulting in a more stable temperature compensation of circadian period over a wider temperature range.

  8. Quantitative Circadian Phosphoproteomic Analysis of Arabidopsis Reveals Extensive Clock Control of Key Components in Physiological, Metabolic, and Signaling Pathways*

    PubMed Central

    Choudhary, Mani Kant; Nomura, Yuko; Wang, Lei; Nakagami, Hirofumi; Somers, David E.

    2015-01-01

    The circadian clock provides adaptive advantages to an organism, resulting in increased fitness and survival. The phosphorylation events that regulate circadian-dependent signaling and the processes which post-translationally respond to clock-gated signals are largely unknown. To better elucidate post-translational events tied to the circadian system we carried out a survey of circadian-regulated protein phosphorylation events in Arabidopsis seedlings. A large-scale mass spectrometry-based quantitative phosphoproteomics approach employing TiO2-based phosphopeptide enrichment techniques identified and quantified 1586 phosphopeptides on 1080 protein groups. A total of 102 phosphopeptides displayed significant changes in abundance, enabling the identification of specific patterns of response to circadian rhythms. Our approach was sensitive enough to quantitate oscillations in the phosphorylation of low abundance clock proteins (EARLY FLOWERING4; ELF4 and PSEUDORESPONSE REGULATOR3; PRR3) as well as other transcription factors and kinases. During constant light, extensive cyclic changes in phosphorylation status occurred in critical regulators, implicating direct or indirect regulation by the circadian system. These included proteins influencing transcriptional regulation, translation, metabolism, stress and phytohormones-mediated responses. We validated our analysis using the elf4–211 allele, in which an S45L transition removes the phosphorylation herein identified. We show that removal of this phosphorylatable site diminishes interaction with EARLY FLOWERING3 (ELF3), a key partner in a tripartite evening complex required for circadian cycling. elf4–211 lengthens period, which increases with increasing temperature, relative to the wild type, resulting in a more stable temperature compensation of circadian period over a wider temperature range. PMID:26091701

  9. Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action.

    PubMed

    Wang, Pengcheng; Xue, Liang; Batelli, Giorgia; Lee, Shinyoung; Hou, Yueh-Ju; Van Oosten, Michael J; Zhang, Huiming; Tao, W Andy; Zhu, Jian-Kang

    2013-07-01

    Sucrose nonfermenting 1 (SNF1)-related protein kinase 2s (SnRK2s) are central components of abscisic acid (ABA) signaling pathways. The snrk2.2/2.3/2.6 triple-mutant plants are nearly completely insensitive to ABA, suggesting that most of the molecular actions of ABA are triggered by the SnRK2s-mediated phosphorylation of substrate proteins. Only a few substrate proteins of the SnRK2s are known. To identify additional substrate proteins of the SnRK2s and provide insight into the molecular actions of ABA, we used quantitative phosphoproteomics to compare the global changes in phosphopeptides in WT and snrk2.2/2.3/2.6 triple mutant seedlings in response to ABA treatment. Among the 5,386 unique phosphorylated peptides identified in this study, we found that ABA can increase the phosphorylation of 166 peptides and decrease the phosphorylation of 117 peptides in WT seedlings. In the snrk2.2/2.3/2.6 triple mutant, 84 of the 166 peptides, representing 58 proteins, could not be phosphorylated, or phosphorylation was not increased under ABA treatment. In vitro kinase assays suggest that most of the 58 proteins can serve as substrates of the SnRK2s. The SnRK2 substrates include proteins involved in flowering time regulation, RNA and DNA binding, miRNA and epigenetic regulation, signal transduction, chloroplast function, and many other cellular processes. Consistent with the SnRK2 phosphorylation of flowering time regulators, the snrk2.2/2.3/2.6 triple mutant flowered significantly earlier than WT. These results shed new light on the role of the SnRK2 protein kinases and on the downstream effectors of ABA action, and improve our understanding of plant responses to adverse environments.

  10. Quantitative phosphoproteomics unravels biased phosphorylation of serotonin 2A receptor at Ser280 by hallucinogenic versus nonhallucinogenic agonists.

    PubMed

    Karaki, Samah; Becamel, Carine; Murat, Samy; Mannoury la Cour, Clotilde; Millan, Mark J; Prézeau, Laurent; Bockaert, Joël; Marin, Philippe; Vandermoere, Franck

    2014-05-01

    The serotonin 5-HT(2A) receptor is a primary target of psychedelic hallucinogens such as lysergic acid diethylamine, mescaline, and psilocybin, which reproduce some of the core symptoms of schizophrenia. An incompletely resolved paradox is that only some 5-HT(2A) receptor agonists exhibit hallucinogenic activity, whereas structurally related agonists with comparable affinity and activity lack such a psychoactive activity. Using a strategy combining stable isotope labeling by amino acids in cell culture with enrichment in phosphorylated peptides by means of hydrophilic interaction liquid chromatography followed by immobilized metal affinity chromatography, we compared the phosphoproteome in HEK-293 cells transiently expressing the 5-HT(2A) receptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) or the nonhallucinogenic 5-HT(2A) agonist lisuride. Among the 5995 identified phosphorylated peptides, 16 sites were differentially phosphorylated upon exposure of cells to DOI versus lisuride. These include a serine (Ser(280)) located in the third intracellular loop of the 5-HT(2A) receptor, a region important for its desensitization. The specific phosphorylation of Ser(280) by hallucinogens was further validated by quantitative mass spectrometry analysis of immunopurified receptor digests and by Western blotting using a phosphosite specific antibody. The administration of DOI, but not of lisuride, to mice, enhanced the phosphorylation of 5-HT(2A) receptors at Ser(280) in the prefrontal cortex. Moreover, hallucinogens induced a less pronounced desensitization of receptor-operated signaling in HEK-293 cells and neurons than did nonhallucinogenic agonists. The mutation of Ser(280) to aspartic acid (to mimic phosphorylation) reduced receptor desensitization by nonhallucinogenic agonists, whereas its mutation to alanine increased the ability of hallucinogens to desensitize the receptor. This study reveals a biased

  11. Quantitative Phosphoproteomics Unravels Biased Phosphorylation of Serotonin 2A Receptor at Ser280 by Hallucinogenic versus Nonhallucinogenic Agonists*

    PubMed Central

    Karaki, Samah; Becamel, Carine; Murat, Samy; Mannoury la Cour, Clotilde; Millan, Mark J.; Prézeau, Laurent; Bockaert, Joël; Marin, Philippe; Vandermoere, Franck

    2014-01-01

    The serotonin 5-HT2A receptor is a primary target of psychedelic hallucinogens such as lysergic acid diethylamine, mescaline, and psilocybin, which reproduce some of the core symptoms of schizophrenia. An incompletely resolved paradox is that only some 5-HT2A receptor agonists exhibit hallucinogenic activity, whereas structurally related agonists with comparable affinity and activity lack such a psychoactive activity. Using a strategy combining stable isotope labeling by amino acids in cell culture with enrichment in phosphorylated peptides by means of hydrophilic interaction liquid chromatography followed by immobilized metal affinity chromatography, we compared the phosphoproteome in HEK-293 cells transiently expressing the 5-HT2A receptor and exposed to either vehicle or the synthetic hallucinogen 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI) or the nonhallucinogenic 5-HT2A agonist lisuride. Among the 5995 identified phosphorylated peptides, 16 sites were differentially phosphorylated upon exposure of cells to DOI versus lisuride. These include a serine (Ser280) located in the third intracellular loop of the 5-HT2A receptor, a region important for its desensitization. The specific phosphorylation of Ser280 by hallucinogens was further validated by quantitative mass spectrometry analysis of immunopurified receptor digests and by Western blotting using a phosphosite specific antibody. The administration of DOI, but not of lisuride, to mice, enhanced the phosphorylation of 5-HT2A receptors at Ser280 in the prefrontal cortex. Moreover, hallucinogens induced a less pronounced desensitization of receptor-operated signaling in HEK-293 cells and neurons than did nonhallucinogenic agonists. The mutation of Ser280 to aspartic acid (to mimic phosphorylation) reduced receptor desensitization by nonhallucinogenic agonists, whereas its mutation to alanine increased the ability of hallucinogens to desensitize the receptor. This study reveals a biased phosphorylation of

  12. Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action

    PubMed Central

    Wang, Pengcheng; Xue, Liang; Batelli, Giorgia; Lee, Shinyoung; Hou, Yueh-Ju; Van Oosten, Michael J.; Zhang, Huiming; Tao, W. Andy; Zhu, Jian-Kang

    2013-01-01

    Sucrose nonfermenting 1 (SNF1)-related protein kinase 2s (SnRK2s) are central components of abscisic acid (ABA) signaling pathways. The snrk2.2/2.3/2.6 triple-mutant plants are nearly completely insensitive to ABA, suggesting that most of the molecular actions of ABA are triggered by the SnRK2s-mediated phosphorylation of substrate proteins. Only a few substrate proteins of the SnRK2s are known. To identify additional substrate proteins of the SnRK2s and provide insight into the molecular actions of ABA, we used quantitative phosphoproteomics to compare the global changes in phosphopeptides in WT and snrk2.2/2.3/2.6 triple mutant seedlings in response to ABA treatment. Among the 5,386 unique phosphorylated peptides identified in this study, we found that ABA can increase the phosphorylation of 166 peptides and decrease the phosphorylation of 117 peptides in WT seedlings. In the snrk2.2/2.3/2.6 triple mutant, 84 of the 166 peptides, representing 58 proteins, could not be phosphorylated, or phosphorylation was not increased under ABA treatment. In vitro kinase assays suggest that most of the 58 proteins can serve as substrates of the SnRK2s. The SnRK2 substrates include proteins involved in flowering time regulation, RNA and DNA binding, miRNA and epigenetic regulation, signal transduction, chloroplast function, and many other cellular processes. Consistent with the SnRK2 phosphorylation of flowering time regulators, the snrk2.2/2.3/2.6 triple mutant flowered significantly earlier than WT. These results shed new light on the role of the SnRK2 protein kinases and on the downstream effectors of ABA action, and improve our understanding of plant responses to adverse environments. PMID:23776212

  13. Quantitative Phosphoproteome Analysis of Lysophosphatidic Acid Induced Chemotaxis applying Dual-step ¹⁸O Labeling Coupled with Immobilized Metal-ion Affinity Chromatography

    SciTech Connect

    Ding, Shi-Jian; Wang, Yingchun; Jacobs, Jon M.; Qian, Weijun; Yang, Feng; Tolmachev, Aleksey V.; Du, Xiuxia; Wang, Wei; Moore, Ronald J.; Monroe, Matthew E.; Purvine, Samuel O.; Waters, Katrina M.; Heibeck, Tyler H.; Adkins, Joshua N.; Camp, David G.; Klemke, Richard L.; Smith, Richard D.

    2008-10-01

    Reversible protein phosphorylation is a central cellular regulatory mechanism in modulating protein activity and propagating signals within cellular pathways and networks. Development of more effective methods for the simultaneous identification of phosphorylation sites and quantification of temporal changes in protein phosphorylation could provide important insights into molecular signaling mechanisms in a variety of different cellular processes. Here we present an integrated quantitative phosphoproteomics approach and its applications for comparative analysis of Cos-7 cells in response to lysophosphatidic acid (LPA) gradient stimulation. The approach combines trypsin-catalyzed 16O/18O labeling plus 16O/18O-methanol esterification labeling for quantitation, a macro- Immobilized Metal-ion Affinity Chromatography trap for phosphopeptide enrichment, and a monolithic capillary column with integrated electrospray emitter. LC separation and MS/MS is followed by neutral loss-dependent MS/MS/MS for phosphopeptide identification using a linear ion trap (LTQ)-FT mass spectrometer and complementary searching algorithms for interpreting MS/MS spectra. Protein phosphorylation involved in various signaling pathways of cell migration were identified and quantified, such as mitogen-activated protein kinase 1, dual-specificity mitogen-activated protein kinase kinase 2, and dual-specificity tyrosine-phosphorylation regulated kinase 1b, and a number of Rho GTPase-activating proteins. These results demonstrate the efficiency of this quantitative phosphoproteomics approach and its application for rapid discovery of phosphorylation events associated with gradient sensing and cell chemotaxis.

  14. Quantitative trait locus analysis of leaf dissection in tomato using Lycopersicon pennellii segmental introgression lines.

    PubMed Central

    Holtan, Hans E E; Hake, Sarah

    2003-01-01

    Leaves are one of the most conspicuous and important organs of all seed plants. A fundamental source of morphological diversity in leaves is the degree to which the leaf is dissected by lobes and leaflets. We used publicly available segmental introgression lines to describe the quantitative trait loci (QTL) controlling the difference in leaf dissection seen between two tomato species, Lycopersicon esculentum and L. pennellii. We define eight morphological characteristics that comprise the mature tomato leaf and describe loci that affect each of these characters. We found 30 QTL that contribute one or more of these characters. Of these 30 QTL, 22 primarily affect leaf dissection and 8 primarily affect leaf size. On the basis of which characters are affected, four classes of loci emerge that affect leaf dissection. The majority of the QTL produce phenotypes intermediate to the two parent lines, while 5 QTL result in transgression with drastically increased dissection relative to both parent lines. PMID:14668401

  15. Quantitative trait locus analysis of leaf dissection in tomato using Lycopersicon pennellii segmental introgression lines.

    PubMed

    Holtan, Hans E E; Hake, Sarah

    2003-11-01

    Leaves are one of the most conspicuous and important organs of all seed plants. A fundamental source of morphological diversity in leaves is the degree to which the leaf is dissected by lobes and leaflets. We used publicly available segmental introgression lines to describe the quantitative trait loci (QTL) controlling the difference in leaf dissection seen between two tomato species, Lycopersicon esculentum and L. pennellii. We define eight morphological characteristics that comprise the mature tomato leaf and describe loci that affect each of these characters. We found 30 QTL that contribute one or more of these characters. Of these 30 QTL, 22 primarily affect leaf dissection and 8 primarily affect leaf size. On the basis of which characters are affected, four classes of loci emerge that affect leaf dissection. The majority of the QTL produce phenotypes intermediate to the two parent lines, while 5 QTL result in transgression with drastically increased dissection relative to both parent lines. PMID:14668401

  16. Quantitative Site-Specific Phosphoproteomics of Trichoderma reesei Signaling Pathways upon Induction of Hydrolytic Enzyme Production.

    PubMed

    Nguyen, Elizabeth V; Imanishi, Susumu Y; Haapaniemi, Pekka; Yadav, Avinash; Saloheimo, Markku; Corthals, Garry L; Pakula, Tiina M

    2016-02-01

    The filamentous fungus Trichoderma reesei is used for industrial production of secreted enzymes including carbohydrate active enzymes, such as cellulases and hemicellulases. The production of many of these enzymes by T. reesei is influenced by the carbon source it grows on, where the regulation system controlling hydrolase genes involves various signaling pathways. T. reesei was cultivated in the presence of sorbitol, a carbon source that does not induce the production of cellulases and hemicellulases, and then exposed to either sophorose or spent-grain extract, which are efficient inducers of the enzyme production. Specific changes at phosphorylation sites were investigated in relation to the production of cellulases and hemicellulases using an MS-based framework. Proteome-wide phosphorylation following carbon source exchange was investigated in the early stages of induction: 0, 2, 5, and 10 min. The workflow involved sequential trypsin digestion, TiO2 enrichment, and MS analysis using a Q Exactive mass spectrometer. We report on the identification and quantitation of 1721 phosphorylation sites. Investigation of the data revealed a complex signaling network activated upon induction involving components related to light-mediated cellulase induction, osmoregulation, and carbon sensing. Changes in protein phosphorylation were detected in the glycolytic pathway, suggesting an inhibition of glucose catabolism at 10 min after the addition of sophorose and as early as 2 min after the addition of spent-grain extract. Differential phosphorylation of factors related to carbon storage, intracellular trafficking, cytoskeleton, and cellulase gene regulation were also observed.

  17. Targeted quantitative phosphoproteomic analysis of erythrocyte membranes during blood bank storage.

    PubMed

    Rinalducci, Sara; Longo, Valentina; Ceci, Luigi R; Zolla, Lello

    2015-02-01

    One of the hallmarks of blood bank stored red blood cells (RBCs) is the irreversible transition from a discoid to a spherocyte-like morphology with membrane perturbation and cytoskeleton disorders. Therefore, identification of the storage-associated modifications in the protein-protein interactions between the cytoskeleton and the lipid bilayer may contribute to enlighten the molecular mechanisms involved in the alterations of mechanical properties of stored RBCs. Here we report the results obtained analyzing RBCs after 0, 21 and 35 days of storage under standard blood banking conditions by label free mass spectrometry (MS)-based experiments. We could quantitatively measure changes in the phosphorylation level of crucial phosphopeptides belonging to β-spectrin, ankyrin-1, α-adducin, dematin, glycophorin A and glycophorin C proteins. Data have been validated by both western blotting and pseudo-Multiple Reaction Monitoring (MRM). Although each phosphopeptide showed a distinctive trend, a sharp increase in the phosphorylation level during the storage duration was observed. Phosphopeptide mapping and structural modeling analysis indicated that the phosphorylated residues localize in protein functional domains fundamental for the maintenance of membrane structural integrity. Along with previous morphological evidence acquired by electron microscopy, our results seem to indicate that 21-day storage may represent a key point for the molecular processes leading to the erythrocyte deformability reduction observed during blood storage. These findings could therefore be helpful in understanding and preventing the morphology-linked mechanisms responsible for the post-transfusion survival of preserved RBCs.

  18. Quantitative label-free phosphoproteomics of six different life stages of the late blight pathogen Phytophthora infestans reveals abundant phosphorylation of members of the CRN effector family.

    PubMed

    Resjö, Svante; Ali, Ashfaq; Meijer, Harold J G; Seidl, Michael F; Snel, Berend; Sandin, Marianne; Levander, Fredrik; Govers, Francine; Andreasson, Erik

    2014-04-01

    The oomycete Phytophthora infestans is the causal agent of late blight in potato and tomato. Since the underlying processes that govern pathogenicity and development in P. infestans are largely unknown, we have performed a large-scale phosphoproteomics study of six different P. infestans life stages. We have obtained quantitative data for 2922 phosphopeptides and compared their abundance. Life-stage-specific phosphopeptides include ATP-binding cassette transporters and a kinase that only occurs in appressoria. In an extended data set, we identified 2179 phosphorylation sites and deduced 22 phosphomotifs. Several of the phosphomotifs matched consensus sequences of kinases that occur in P. infestans but not Arabidopsis. In addition, we detected tyrosine phosphopeptides that are potential targets of kinases resembling mammalian tyrosine kinases. Among the phosphorylated proteins are members of the RXLR and Crinkler effector families. The latter are phosphorylated in several life stages and at multiple positions, in sites that are conserved between different members of the Crinkler family. This indicates that proteins in the Crinkler family have functions beyond their putative role as (necrosis-inducing) effectors. This phosphoproteomics data will be instrumental for studies on oomycetes and host-oomycete interactions. The data sets have been deposited to ProteomeXchange (identifier PXD000433).

  19. Quantitative Phosphoproteome Analysis of Bacillus subtilis Reveals Novel Substrates of the Kinase PrkC and Phosphatase PrpC*

    PubMed Central

    Ravikumar, Vaishnavi; Shi, Lei; Krug, Karsten; Derouiche, Abderahmane; Jers, Carsten; Cousin, Charlotte; Kobir, Ahasanul; Mijakovic, Ivan; Macek, Boris

    2014-01-01

    Reversible protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) residues plays a critical role in regulation of vital processes in the cell. Despite of considerable progress in our understanding of the role of this modification in bacterial physiology, the dynamics of protein phosphorylation during bacterial growth has rarely been systematically addressed. In addition, little is known about in vivo substrates of bacterial Ser/Thr/Tyr kinases and phosphatases. An excellent candidate to study these questions is the Gram-positive bacterium Bacillus subtilis, one of the most intensively investigated bacterial model organism with both research and industrial applications. Here we employed gel-free phosphoproteomics combined with SILAC labeling and high resolution mass spectrometry to study the proteome and phosphoproteome dynamics during the batch growth of B. subtilis. We measured the dynamics of 1666 proteins and 64 phosphorylation sites in five distinct phases of growth. Enzymes of the central carbon metabolism and components of the translation machinery appear to be highly phosphorylated in the stationary phase, coinciding with stronger expression of Ser/Thr kinases. We further used the SILAC workflow to identify novel putative substrates of the Ser/Thr kinase PrkC and the phosphatase PrpC during stationary phase. The overall number of putative substrates was low, pointing to a high kinase and phosphatase specificity. One of the phosphorylation sites affected by both, PrkC and PrpC, was the Ser281 on the oxidoreductase YkwC. We showed that PrkC phosphorylates and PrpC dephosphorylates YkwC in vitro and that phosphorylation at Ser281 abolishes the oxidoreductase activity of YkwC in vitro and in vivo. Our results present the most detailed phosphoproteomic analysis of B. subtilis growth to date and provide the first global in vivo screen of PrkC and PrpC substrates. PMID:24390483

  20. Label-free quantitative phosphoproteomics with novel pairwise abundance normalization reveals synergistic RAS and CIP2A signaling.

    PubMed

    Kauko, Otto; Laajala, Teemu Daniel; Jumppanen, Mikael; Hintsanen, Petteri; Suni, Veronika; Haapaniemi, Pekka; Corthals, Garry; Aittokallio, Tero; Westermarck, Jukka; Imanishi, Susumu Y

    2015-08-17

    Hyperactivated RAS drives progression of many human malignancies. However, oncogenic activity of RAS is dependent on simultaneous inactivation of protein phosphatase 2A (PP2A) activity. Although PP2A is known to regulate some of the RAS effector pathways, it has not been systematically assessed how these proteins functionally interact. Here we have analyzed phosphoproteomes regulated by either RAS or PP2A, by phosphopeptide enrichment followed by mass-spectrometry-based label-free quantification. To allow data normalization in situations where depletion of RAS or PP2A inhibitor CIP2A causes a large uni-directional change in the phosphopeptide abundance, we developed a novel normalization strategy, named pairwise normalization. This normalization is based on adjusting phosphopeptide abundances measured before and after the enrichment. The superior performance of the pairwise normalization was verified by various independent methods. Additionally, we demonstrate how the selected normalization method influences the downstream analyses and interpretation of pathway activities. Consequently, bioinformatics analysis of RAS and CIP2A regulated phosphoproteomes revealed a significant overlap in their functional pathways. This is most likely biologically meaningful as we observed a synergistic survival effect between CIP2A and RAS expression as well as KRAS activating mutations in TCGA pan-cancer data set, and synergistic relationship between CIP2A and KRAS depletion in colony growth assays.

  1. Quantitative dissection of the simple repression input–output function

    PubMed Central

    Garcia, Hernan G.; Phillips, Rob

    2011-01-01

    We present a quantitative case study of transcriptional regulation in which we carry out a systematic dialogue between theory and measurement for an important and ubiquitous regulatory motif in bacteria, namely, that of simple repression. This architecture is realized by a single repressor binding site overlapping the promoter. From the theory point of view, this motif is described by a single gene regulation function based upon only a few parameters that are convenient theoretically and accessible experimentally. The usual approach is turned on its side by using the mathematical description of these regulatory motifs as a predictive tool to determine the number of repressors in a collection of strains with a large variation in repressor copy number. The predictions and corresponding measurements are carried out over a large dynamic range in both expression fold change (spanning nearly four orders of magnitude) and repressor copy number (spanning about two orders of magnitude). The predictions are tested by measuring the resulting level of gene expression and are then validated by using quantitative immunoblots. The key outcomes of this study include a systematic quantitative analysis of the limits and validity of the input–output relation for simple repression, a precise determination of the in vivo binding energies for DNA–repressor interactions for several distinct repressor binding sites, and a repressor census for Lac repressor in Escherichia coli. PMID:21730194

  2. Quantitative Phosphoproteomics of the Ataxia Telangiectasia-Mutated (ATM) and Ataxia Telangiectasia-Mutated and Rad3-related (ATR) Dependent DNA Damage Response in Arabidopsis thaliana*

    PubMed Central

    Roitinger, Elisabeth; Hofer, Manuel; Köcher, Thomas; Pichler, Peter; Novatchkova, Maria; Yang, Jianhua; Schlögelhofer, Peter; Mechtler, Karl

    2015-01-01

    The reversible phosphorylation of proteins on serine, threonine, and tyrosine residues is an important biological regulatory mechanism. In the context of genome integrity, signaling cascades driven by phosphorylation are crucial for the coordination and regulation of DNA repair. The two serine/threonine protein kinases ataxia telangiectasia-mutated (ATM) and Ataxia telangiectasia-mutated and Rad3-related (ATR) are key factors in this process, each specific for different kinds of DNA lesions. They are conserved across eukaryotes, mediating the activation of cell-cycle checkpoints, chromatin modifications, and regulation of DNA repair proteins. We designed a novel mass spectrometry-based phosphoproteomics approach to study DNA damage repair in Arabidopsis thaliana. The protocol combines filter aided sample preparation, immobilized metal affinity chromatography, metal oxide affinity chromatography, and strong cation exchange chromatography for phosphopeptide generation, enrichment, and separation. Isobaric labeling employing iTRAQ (isobaric tags for relative and absolute quantitation) was used for profiling the phosphoproteome of atm atr double mutants and wild type plants under either regular growth conditions or challenged by irradiation. A total of 10,831 proteins were identified and 15,445 unique phosphopeptides were quantified, containing 134 up- and 38 down-regulated ATM/ATR dependent phosphopeptides. We identified known and novel ATM/ATR targets such as LIG4 and MRE11 (needed for resistance against ionizing radiation), PIE1 and SDG26 (implicated in chromatin remodeling), PCNA1, WAPL, and PDS5 (implicated in DNA replication), and ASK1 and HTA10 (involved in meiosis). PMID:25561503

  3. Quantitative expression proteomics and phosphoproteomics profile of brain from PINK1 knockout mice: insights into mechanisms of familial Parkinson's disease.

    PubMed

    Triplett, Judy C; Zhang, Zhaoshu; Sultana, Rukhsana; Cai, Jian; Klein, Jon B; Büeler, Hansruedi; Butterfield, David Allan

    2015-06-01

    Parkinson's disease (PD) is an age-related, neurodegenerative motor disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta and presence of α-synuclein-containing protein aggregates. Mutations in the mitochondrial Ser/Thr kinase PTEN-induced kinase 1 (PINK1) are associated with an autosomal recessive familial form of early-onset PD. Recent studies have suggested that PINK1 plays important neuroprotective roles against mitochondrial dysfunction by phosphorylating and recruiting Parkin, a cytosolic E3 ubiquitin ligase, to facilitate elimination of damaged mitochondria via autophagy-lysosomal pathways. Loss of PINK1 in cells and animals leads to various mitochondrial impairments and oxidative stress, culminating in dopaminergic neuronal death in humans. Using a 2-D polyacrylamide gel electrophoresis proteomics approach, the differences in expressed brain proteome and phosphoproteome between 6-month-old PINK1-deficient mice and wild-type mice were identified. The observed changes in the brain proteome and phosphoproteome of mice lacking PINK1 suggest that defects in signaling networks, energy metabolism, cellular proteostasis, and neuronal structure and plasticity are involved in the pathogenesis of familial PD. Mutations in PINK1 are associated with an early-onset form of Parkinson's disease (PD). This study examines changes in the proteome and phosphoproteome of the PINK1 knockout mouse brain. Alterations were noted in several key proteins associated with: increased oxidative stress, aberrant cellular signaling, altered neuronal structure, decreased synaptic plasticity, reduced neurotransmission, diminished proteostasis networks, and altered metabolism. 14-3-3ε, 14-3-3 protein epsilon; 3-PGDH, phosphoglycerate dehydrogenase; ALDOA, aldolase A; APT1, acyl-protein thioesterase 1; CaM, calmodulin; CBR3, carbonyl reductase [NADPH] 3; ENO2, gamma-enolase; HPRT, hypoxanthine-guanine phosphoribosyltransferase; HSP70

  4. Dissecting Dissection.

    ERIC Educational Resources Information Center

    AV Magazine, 1996

    1996-01-01

    This journal features articles covering various aspects of dissection. "Biology--The Study of Life" (George Russell) offers students experiments that do not require using invasive procedures. "Animal Cruelty--Behind the Scenes" (Zoe Weil) describes sources of laboratory animals. "Doing without Dissection" (Juliana Texley) discusses objections over…

  5. Quantitative phosphoproteomics reveals genistein as a modulator of cell cycle and DNA damage response pathways in triple-negative breast cancer cells

    PubMed Central

    FANG, YI; ZHANG, QIAN; WANG, XIN; YANG, XUE; WANG, XIANGYU; HUANG, ZHEN; JIAO, YUCHEN; WANG, JING

    2016-01-01

    Around one sixth of breast cancer cases are classified as triple-negative breast cancer (TNBC), named after the absence of the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2); however, patients with TNBC suffer from poor clinical outcome and shortage of targeted therapy. Genistein, an estrogenic soy isoflavone, shows anticancer effects in TNBC cells such as inducing G2/M cell cycle arrest and apoptosis. However, the underlying mechanism of its anticancer effects is poorly understood and its elucidation can help the development of novel therapeutic strategies for TNBC. In this study, by combining isobaric tag-based TMT labeling with titanium dioxide-based phosphopeptide enrichment, we quantitated 5,445 phosphorylation sites on 2,008 phosphoproteins in the TNBC cell line MDA-MB-231, upon genistein treatment. Our analysis revealed 332 genistein-regulated phosphorylation sites on 226 proteins. Our data show that genistein can regulate several biological processes during the cell cycle, including DNA replication, cohesin complex cleavage, and kinetochore formation. Furthermore, genistein can also activate DNA damage response, including activation of ATR and BRCA1 complex. Overall, our study presents evidence at a phosphoproteomic level that genistein is able to inhibit TNBC cell growth by regulating the cell cycle and DNA damage response in a more complex manner. Our findings help elucidate the mechanisms through which genistein exerts its anticancer effects in TNBC cells. PMID:26783066

  6. Quantitative Phosphoproteomics Analysis Reveals a Key Role of Insulin Growth Factor 1 Receptor (IGF1R) Tyrosine Kinase in Human Sperm Capacitation*

    PubMed Central

    Wang, Jing; Qi, Lin; Huang, Shaoping; Zhou, Tao; Guo, Yueshuai; Wang, Gaigai; Guo, Xuejiang; Zhou, Zuomin; Sha, Jiahao

    2015-01-01

    One of the most important changes during sperm capacitation is the enhancement of tyrosine phosphorylation. However, the mechanisms of protein tyrosine phosphorylation during sperm capacitation are not well studied. We used label-free quantitative phosphoproteomics to investigate the overall phosphorylation events during sperm capacitation in humans and identified 231 sites with increased phosphorylation levels. Motif analysis using the NetworKIN algorithm revealed that the activity of tyrosine phosphorylation kinases insulin growth factor 1 receptor (IGF1R)/insulin receptor is significantly enriched among the up-regulated phosphorylation substrates during capacitation. Western blotting further confirmed inhibition of IGF1R with inhibitors GSK1904529A and NVP-AEW541, which inhibited the increase in tyrosine phosphorylation levels during sperm capacitation. Additionally, sperm hyperactivated motility was also inhibited by GSK1904529A and NVP-AEW541 but could be up-regulated by insulin growth factor 1, the ligand of IGF1R. Thus, the IGF1R-mediated tyrosine phosphorylation pathway may play important roles in the regulation of sperm capacitation in humans and could be a target for improvement in sperm functions in infertile men. PMID:25693802

  7. Quantitative phosphoproteomics analysis reveals a key role of insulin growth factor 1 receptor (IGF1R) tyrosine kinase in human sperm capacitation.

    PubMed

    Wang, Jing; Qi, Lin; Huang, Shaoping; Zhou, Tao; Guo, Yueshuai; Wang, Gaigai; Guo, Xuejiang; Zhou, Zuomin; Sha, Jiahao

    2015-04-01

    One of the most important changes during sperm capacitation is the enhancement of tyrosine phosphorylation. However, the mechanisms of protein tyrosine phosphorylation during sperm capacitation are not well studied. We used label-free quantitative phosphoproteomics to investigate the overall phosphorylation events during sperm capacitation in humans and identified 231 sites with increased phosphorylation levels. Motif analysis using the NetworKIN algorithm revealed that the activity of tyrosine phosphorylation kinases insulin growth factor 1 receptor (IGF1R)/insulin receptor is significantly enriched among the up-regulated phosphorylation substrates during capacitation. Western blotting further confirmed inhibition of IGF1R with inhibitors GSK1904529A and NVP-AEW541, which inhibited the increase in tyrosine phosphorylation levels during sperm capacitation. Additionally, sperm hyperactivated motility was also inhibited by GSK1904529A and NVP-AEW541 but could be up-regulated by insulin growth factor 1, the ligand of IGF1R. Thus, the IGF1R-mediated tyrosine phosphorylation pathway may play important roles in the regulation of sperm capacitation in humans and could be a target for improvement in sperm functions in infertile men.

  8. SILAC for global phosphoproteomic analysis.

    PubMed

    Pimienta, Genaro; Chaerkady, Raghothama; Pandey, Akhilesh

    2009-01-01

    Establishing the phosphorylation pattern of proteins in a comprehensive fashion is an important goal of a majority of cell signaling projects. Phosphoproteomic strategies should be designed in such a manner as to identify sites of phosphorylation as well as to provide quantitative information about the extent of phosphorylation at the sites. In this chapter, we describe an experimental strategy that outlines such an approach using stable isotope labeling with amino acids in cell culture (SILAC) coupled to LC-MS/MS. We highlight the importance of quantitative strategies in signal transduction as a platform for a systematic and global elucidation of biological processes.

  9. Integrated genomics and molecular breeding approaches for dissecting the complex quantitative traits in crop plants.

    PubMed

    Kujur, Alice; Saxena, Maneesha S; Bajaj, Deepak; Laxmi; Parida, Swarup K

    2013-12-01

    The enormous population growth, climate change and global warming are now considered major threats to agriculture and world's food security. To improve the productivity and sustainability of agriculture, the development of highyielding and durable abiotic and biotic stress-tolerant cultivars and/climate resilient crops is essential. Henceforth, understanding the molecular mechanism and dissection of complex quantitative yield and stress tolerance traits is the prime objective in current agricultural biotechnology research. In recent years, tremendous progress has been made in plant genomics and molecular breeding research pertaining to conventional and next-generation whole genome, transcriptome and epigenome sequencing efforts, generation of huge genomic, transcriptomic and epigenomic resources and development of modern genomics-assisted breeding approaches in diverse crop genotypes with contrasting yield and abiotic stress tolerance traits. Unfortunately, the detailed molecular mechanism and gene regulatory networks controlling such complex quantitative traits is not yet well understood in crop plants. Therefore, we propose an integrated strategies involving available enormous and diverse traditional and modern -omics (structural, functional, comparative and epigenomics) approaches/resources and genomics-assisted breeding methods which agricultural biotechnologist can adopt/utilize to dissect and decode the molecular and gene regulatory networks involved in the complex quantitative yield and stress tolerance traits in crop plants. This would provide clues and much needed inputs for rapid selection of novel functionally relevant molecular tags regulating such complex traits to expedite traditional and modern marker-assisted genetic enhancement studies in target crop species for developing high-yielding stress-tolerant varieties.

  10. Quantitative and Functional Phosphoproteomic Analysis Reveals that Ethylene Regulates Water Transport via the C-Terminal Phosphorylation of Aquaporin PIP2;1 in Arabidopsis.

    PubMed

    Qing, Dongjin; Yang, Zhu; Li, Mingzhe; Wong, Wai Shing; Guo, Guangyu; Liu, Shichang; Guo, Hongwei; Li, Ning

    2016-01-01

    Ethylene participates in the regulation of numerous cellular events and biological processes, including water loss, during leaf and flower petal wilting. The diverse ethylene responses may be regulated via dynamic interplays between protein phosphorylation/dephosphorylation and ubiquitin/26S proteasome-mediated protein degradation and protease cleavage. To address how ethylene alters protein phosphorylation through multi-furcated signaling pathways, we performed a (15)N stable isotope labelling-based, differential, and quantitative phosphoproteomics study on air- and ethylene-treated ethylene-insensitive Arabidopsis double loss-of-function mutant ein3-1/eil1-1. Among 535 non-redundant phosphopeptides identified, two and four phosphopeptides were up- and downregulated by ethylene, respectively. Ethylene-regulated phosphorylation of aquaporin PIP2;1 is positively correlated with the water flux rate and water loss in leaf. Genetic studies in combination with quantitative proteomics, immunoblot analysis, protoplast swelling/shrinking experiments, and leaf water loss assays on the transgenic plants expressing both the wild-type and S280A/S283A-mutated PIP2;1 in the both Col-0 and ein3eil1 genetic backgrounds suggest that ethylene increases water transport rate in Arabidopsis cells by enhancing S280/S283 phosphorylation at the C terminus of PIP2;1. Unknown kinase and/or phosphatase activities may participate in the initial up-regulation independent of the cellular functions of EIN3/EIL1. This finding contributes to our understanding of ethylene-regulated leaf wilting that is commonly observed during post-harvest storage of plant organs.

  11. Quantitative Tools for Dissection of Hydrogen-Producing Metabolic Networks-Final Report

    SciTech Connect

    Rabinowitz, Joshua D.; Dismukes, G.Charles.; Rabitz, Herschel A.; Amador-Noguez, Daniel

    2012-10-19

    During this project we have pioneered the development of integrated experimental-computational technologies for the quantitative dissection of metabolism in hydrogen and biofuel producing microorganisms (i.e. C. acetobutylicum and various cyanobacteria species). The application of these new methodologies resulted in many significant advances in the understanding of the metabolic networks and metabolism of these organisms, and has provided new strategies to enhance their hydrogen or biofuel producing capabilities. As an example, using mass spectrometry, isotope tracers, and quantitative flux-modeling we mapped the metabolic network structure in C. acetobutylicum. This resulted in a comprehensive and quantitative understanding of central carbon metabolism that could not have been obtained using genomic data alone. We discovered that biofuel production in this bacterium, which only occurs during stationary phase, requires a global remodeling of central metabolism (involving large changes in metabolite concentrations and fluxes) that has the effect of redirecting resources (carbon and reducing power) from biomass production into solvent production. This new holistic, quantitative understanding of metabolism is now being used as the basis for metabolic engineering strategies to improve solvent production in this bacterium. In another example, making use of newly developed technologies for monitoring hydrogen and NAD(P)H levels in vivo, we dissected the metabolic pathways for photobiological hydrogen production by cyanobacteria Cyanothece sp. This investigation led to the identification of multiple targets for improving hydrogen production. Importantly, the quantitative tools and approaches that we have developed are broadly applicable and we are now using them to investigate other important biofuel producers, such as cellulolytic bacteria.

  12. Acute Aortic Dissection Biomarkers Identified Using Isobaric Tags for Relative and Absolute Quantitation.

    PubMed

    Xiao, Ziya; Xue, Yuan; Yao, Chenling; Gu, Guorong; Zhang, Yaping; Zhang, Jin; Fan, Fan; Luan, Xiao; Deng, Zhi; Tao, Zhengang; Song, Zhen-Ju; Tong, Chaoyang; Wang, Haojun

    2016-01-01

    The purpose of this study was to evaluate the utility of potential serum biomarkers for acute aortic dissection (AAD) that were identified by isobaric Tags for Relative and Absolute Quantitation (iTRAQ) approaches. Serum samples from 20 AAD patients and 20 healthy volunteers were analyzed using iTRAQ technology. Protein validation was performed using samples from 120 patients with chest pain. A total of 355 proteins were identified with the iTRAQ approach; 164 proteins reached the strict quantitative standard, and 125 proteins were increased or decreased more than 1.2-fold (64 and 61 proteins were up- and downregulated, resp.). Lumican, C-reactive protein (CRP), thrombospondin-1 (TSP-1), and D-dimer were selected as candidate biomarkers for the validation tests. Receiver operating characteristic (ROC) curves show that Lumican and D-dimer have diagnostic value (area under the curves [AUCs] 0.895 and 0.891, P < 0.05). For Lumican, the diagnostic sensitivity and specificity were 73.33% and 98.33%, while the corresponding values for D-dimer were 93.33% and 68.33%. For Lumican and D-dimer AAD combined diagnosis, the sensitivity and specificity were 88.33% and 95%, respectively. In conclusion, Lumican has good specificity and D-dimer has good sensitivity for the diagnosis of AAD, while the combined detection of D-dimer and Lumican has better diagnostic value. PMID:27403433

  13. Acute Aortic Dissection Biomarkers Identified Using Isobaric Tags for Relative and Absolute Quantitation

    PubMed Central

    Xiao, Ziya; Xue, Yuan; Gu, Guorong; Zhang, Yaping; Zhang, Jin; Fan, Fan; Luan, Xiao; Deng, Zhi; Tao, Zhengang; Song, Zhen-ju; Tong, Chaoyang; Wang, Haojun

    2016-01-01

    The purpose of this study was to evaluate the utility of potential serum biomarkers for acute aortic dissection (AAD) that were identified by isobaric Tags for Relative and Absolute Quantitation (iTRAQ) approaches. Serum samples from 20 AAD patients and 20 healthy volunteers were analyzed using iTRAQ technology. Protein validation was performed using samples from 120 patients with chest pain. A total of 355 proteins were identified with the iTRAQ approach; 164 proteins reached the strict quantitative standard, and 125 proteins were increased or decreased more than 1.2-fold (64 and 61 proteins were up- and downregulated, resp.). Lumican, C-reactive protein (CRP), thrombospondin-1 (TSP-1), and D-dimer were selected as candidate biomarkers for the validation tests. Receiver operating characteristic (ROC) curves show that Lumican and D-dimer have diagnostic value (area under the curves [AUCs] 0.895 and 0.891, P < 0.05). For Lumican, the diagnostic sensitivity and specificity were 73.33% and 98.33%, while the corresponding values for D-dimer were 93.33% and 68.33%. For Lumican and D-dimer AAD combined diagnosis, the sensitivity and specificity were 88.33% and 95%, respectively. In conclusion, Lumican has good specificity and D-dimer has good sensitivity for the diagnosis of AAD, while the combined detection of D-dimer and Lumican has better diagnostic value. PMID:27403433

  14. Comprehensive Quantitative Comparison of the Membrane Proteome, Phosphoproteome, and Sialiome of Human Embryonic and Neural Stem Cells*

    PubMed Central

    Melo-Braga, Marcella Nunes; Schulz, Melanie; Liu, Qiuyue; Swistowski, Andrzej; Palmisano, Giuseppe; Engholm-Keller, Kasper; Jakobsen, Lene; Zeng, Xianmin; Larsen, Martin Røssel

    2014-01-01

    Human embryonic stem cells (hESCs) can differentiate into neural stem cells (NSCs), which can further be differentiated into neurons and glia cells. Therefore, these cells have huge potential as source for treatment of neurological diseases. Membrane-associated proteins are very important in cellular signaling and recognition, and their function and activity are frequently regulated by post-translational modifications such as phosphorylation and glycosylation. To obtain information about membrane-associated proteins and their modified amino acids potentially involved in changes of hESCs and NSCs as well as to investigate potential new markers for these two cell stages, we performed large-scale quantitative membrane-proteomic of hESCs and NSCs. This approach employed membrane purification followed by peptide dimethyl labeling and peptide enrichment to study the membrane subproteome as well as changes in phosphorylation and sialylation between hESCs and NSCs. Combining proteomics and modification specific proteomics we identified a total of 5105 proteins whereof 57% contained transmembrane domains or signal peptides. The enrichment strategy yielded a total of 10,087 phosphorylated peptides in which 78% of phosphopeptides were identified with ≥99% confidence in site assignment and 1810 unique formerly sialylated N-linked glycopeptides. Several proteins were identified as significantly regulated in hESCs and NSC, including proteins involved in the early embryonic and neural development. In the latter group of proteins, we could identify potential NSC markers as Crumbs 2 and several novel proteins. A motif analysis of the altered phosphosites showed a sequence consensus motif (R-X-XpS/T) significantly up-regulated in NSC. This motif is among other kinases recognized by the calmodulin-dependent protein kinase-2, emphasizing a possible importance of this kinase for this cell stage. Collectively, this data represent the most diverse set of post

  15. Comprehensive quantitative comparison of the membrane proteome, phosphoproteome, and sialiome of human embryonic and neural stem cells.

    PubMed

    Melo-Braga, Marcella Nunes; Schulz, Melanie; Liu, Qiuyue; Swistowski, Andrzej; Palmisano, Giuseppe; Engholm-Keller, Kasper; Jakobsen, Lene; Zeng, Xianmin; Larsen, Martin Røssel

    2014-01-01

    Human embryonic stem cells (hESCs) can differentiate into neural stem cells (NSCs), which can further be differentiated into neurons and glia cells. Therefore, these cells have huge potential as source for treatment of neurological diseases. Membrane-associated proteins are very important in cellular signaling and recognition, and their function and activity are frequently regulated by post-translational modifications such as phosphorylation and glycosylation. To obtain information about membrane-associated proteins and their modified amino acids potentially involved in changes of hESCs and NSCs as well as to investigate potential new markers for these two cell stages, we performed large-scale quantitative membrane-proteomic of hESCs and NSCs. This approach employed membrane purification followed by peptide dimethyl labeling and peptide enrichment to study the membrane subproteome as well as changes in phosphorylation and sialylation between hESCs and NSCs. Combining proteomics and modification specific proteomics we identified a total of 5105 proteins whereof 57% contained transmembrane domains or signal peptides. The enrichment strategy yielded a total of 10,087 phosphorylated peptides in which 78% of phosphopeptides were identified with ≥99% confidence in site assignment and 1810 unique formerly sialylated N-linked glycopeptides. Several proteins were identified as significantly regulated in hESCs and NSC, including proteins involved in the early embryonic and neural development. In the latter group of proteins, we could identify potential NSC markers as Crumbs 2 and several novel proteins. A motif analysis of the altered phosphosites showed a sequence consensus motif (R-X-XpS/T) significantly up-regulated in NSC. This motif is among other kinases recognized by the calmodulin-dependent protein kinase-2, emphasizing a possible importance of this kinase for this cell stage. Collectively, this data represent the most diverse set of post

  16. Quantitative phosphoproteomic analysis reveals γ-bisabolene inducing p53-mediated apoptosis of human oral squamous cell carcinoma via HDAC2 inhibition and ERK1/2 activation.

    PubMed

    Jou, Yu-Jen; Chen, Chao-Jung; Liu, Yu-Ching; Way, Tzong-Der; Lai, Chih-Ho; Hua, Chun-Hung; Wang, Ching-Ying; Huang, Su-Hua; Kao, Jung-Yie; Lin, Cheng-Wen

    2015-10-01

    γ-Bisabolene, one of main components in cardamom, showed potent in vitro and in vivo anti-proliferative activities against human oral squamous cell carcinoma (OSCC). γ-Bisabolene activated caspases-3/9 and decreased mitochondrial memebrane potential, leading to apoptosis of OSCC cell lines (Ca9-22 and SAS), but not normal oral fibroblast cells. Phosphoproteome profiling of OSCC cells treated with γ-bisabolene was identified using TiO2-PDMS plate and LC-MS/MS, then confirmed using Western blotting and real-time RT-PCR assays. Phosphoproteome profiling revealed that γ-bisabolene increased the phosphorylation of ERK1/2, protein phosphatases 1 (PP1), and p53, as well as decreased the phosphorylation of histone deacetylase 2 (HDAC2) in the process of apoptosis induction. Protein-protein interaction network analysis proposed the involvement of PP1-HDAC2-p53 and ERK1/2-p53 pathways in γ-bisabolene-induced apoptosis. Subsequent assays indicated γ-bisabolene eliciting p53 acetylation that enhanced the expression of p53-regulated apoptotic genes. PP1 inhibitor-2 restored the status of HDAC2 phosphorylation, reducing p53 acetylation and PUMA mRNA expression in γ-bisabolene-treated Ca9-22 and SAS cells. Meanwhile, MEK and ERK inhibitors significantly decreased γ-bisabolene-induced PUMA expression in both cancer cell lines. Notably, the results ascertained the involvement of PP1-HDAC2-p53 and ERK1/2-p53 pathways in mitochondria-mediated apoptosis of γ-bisabolene-treated cells. This study demonstrated γ-bisabolene displaying potent anti-proliferative and apoptosis-inducing activities against OSCC in vitro and in vivo, elucidating molecular mechanisms of γ-bisabolene-induced apoptosis. The novel insight could be useful for developing anti-cancer drugs. PMID:26194454

  17. Quantitative phosphoproteomics of tomato mounting a hypersensitive response reveals a swift suppression of photosynthetic activity and a differential role for hsp90 isoforms.

    PubMed

    Stulemeijer, Iris J E; Joosten, Matthieu H A J; Jensen, Ole N

    2009-03-01

    An important mechanism by which plants defend themselves against pathogens is the rapid execution of a hypersensitive response (HR). Tomato plants containing the Cf-4 resistance gene mount an HR that relies on the activation of phosphorylation cascades, when challenged with the Avr4 elicitor secreted by the pathogenic fungus Cladosporium fulvum. Phosphopeptides were isolated from tomato seedlings expressing both Cf-4 and Avr4 using titanium dioxide columns and LC-MS/MS analysis led to the identification of 50 phosphoproteins, most of which have not been described in tomato before. Phosphopeptides were quantified using a label-free approach based on the MS peak areas. We identified 12 phosphopeptides for which the abundance changed upon HR initiation, as compared to control seedlings. Our results suggest that photosynthetic activity is specifically suppressed in a phosphorylation-dependent way during the very early stages of HR development. In addition, phosphopeptides originating from four Hsp90 isoforms exhibited altered abundances in Cf-4/Avr4 seedlings compared to control seedlings, suggesting that the isoforms of this chaperone protein have a different function in defense signaling. We show that label-free relative quantification of the phosphoproteome of complex samples is feasible, allowing extension of our knowledge on the general physiology and defense signaling of plants mounting the HR. PMID:19178300

  18. Label‐free quantitative analysis of the casein kinase 2‐responsive phosphoproteome of the marine minimal model species Ostreococcus tauri

    PubMed Central

    Le Bihan, Thierry; Hindle, Matthew; Martin, Sarah F.; Barrios‐Llerena, Martin E.; Krahmer, Johanna; Kis, Katalin; Millar, Andrew J.

    2015-01-01

    Casein kinase 2 (CK2) is a protein kinase that phosphorylates a plethora of cellular target proteins involved in processes including DNA repair, cell cycle control, and circadian timekeeping. CK2 is functionally conserved across eukaryotes, although the substrate proteins identified in a range of complex tissues are often different. The marine alga Ostreococcus tauri is a unicellular eukaryotic model organism ideally suited to efficiently study generic roles of CK2 in the cellular circadian clock. Overexpression of CK2 leads to a slow circadian rhythm, verifying functional conservation of CK2 in timekeeping. The proteome was analysed in wild‐type and CK2‐overexpressing algae at dawn and dusk, revealing that differential abundance of the global proteome across the day is largely unaffected by overexpression. However, CK2 activity contributed more strongly to timekeeping at dusk than at dawn. The phosphoproteome of a CK2 overexpression line and cells treated with CK2 inhibitor was therefore analysed and compared to control cells at dusk. We report an extensive catalogue of 447 unique CK2‐responsive differential phosphopeptide motifs to inform future studies into CK2 activity in the circadian clock of more complex tissues. All MS data have been deposited in the ProteomeXchange with identifier PXD000975 (http://proteomecentral.proteomexchange.org/dataset/PXD000975). PMID:25930153

  19. Stable Isotope Metabolic Labeling-based Quantitative Phosphoproteomic Analysis of Arabidopsis Mutants Reveals Ethylene-regulated Time-dependent Phosphoproteins and Putative Substrates of Constitutive Triple Response 1 Kinase*

    PubMed Central

    Yang, Zhu; Guo, Guangyu; Zhang, Manyu; Liu, Claire Y.; Hu, Qin; Lam, Henry; Cheng, Han; Xue, Yu; Li, Jiayang; Li, Ning

    2013-01-01

    Ethylene is an important plant hormone that regulates numerous cellular processes and stress responses. The mode of action of ethylene is both dose- and time-dependent. Protein phosphorylation plays a key role in ethylene signaling, which is mediated by the activities of ethylene receptors, constitutive triple response 1 (CTR1) kinase, and phosphatase. To address how ethylene alters the cellular protein phosphorylation profile in a time-dependent manner, differential and quantitative phosphoproteomics based on 15N stable isotope labeling in Arabidopsis was performed on both one-minute ethylene-treated Arabidopsis ethylene-overly-sensitive loss-of-function mutant rcn1-1, deficient in PP2A phosphatase activity, and a pair of long-term ethylene-treated wild-type and loss-of-function ethylene signaling ctr1-1 mutants, deficient in mitogen-activated kinase kinase kinase activity. In total, 1079 phosphopeptides were identified, among which 44 were novel. Several one-minute ethylene-regulated phosphoproteins were found from the rcn1-1. Bioinformatic analysis of the rcn1-1 phosphoproteome predicted nine phosphoproteins as the putative substrates for PP2A phosphatase. In addition, from CTR1 kinase-enhanced phosphosites, we also found putative CTR1 kinase substrates including plastid transcriptionally active protein and calcium-sensing receptor. These regulatory proteins are phosphorylated in the presence of ethylene. Analysis of ethylene-regulated phosphosites using the group-based prediction system with a protein–protein interaction filter revealed a total of 14 kinase–substrate relationships that may function in both CTR1 kinase- and PP2A phosphatase-mediated phosphor-relay pathways. Finally, several ethylene-regulated post-translational modification network models have been built using molecular systems biology tools. It is proposed that ethylene regulates the phosphorylation of arginine/serine-rich splicing factor 41, plasma membrane intrinsic protein 2A, light

  20. Quantitative phosphoproteomics reveals the role of the AMPK plant ortholog SnRK1 as a metabolic master regulator under energy deprivation

    PubMed Central

    Nukarinen, Ella; Nägele, Thomas; Pedrotti, Lorenzo; Wurzinger, Bernhard; Mair, Andrea; Landgraf, Ramona; Börnke, Frederik; Hanson, Johannes; Teige, Markus; Baena-Gonzalez, Elena; Dröge-Laser, Wolfgang; Weckwerth, Wolfram

    2016-01-01

    Since years, research on SnRK1, the major cellular energy sensor in plants, has tried to define its role in energy signalling. However, these attempts were notoriously hampered by the lethality of a complete knockout of SnRK1. Therefore, we generated an inducible amiRNA::SnRK1α2 in a snrk1α1 knock out background (snrk1α1/α2) to abolish SnRK1 activity to understand major systemic functions of SnRK1 signalling under energy deprivation triggered by extended night treatment. We analysed the in vivo phosphoproteome, proteome and metabolome and found that activation of SnRK1 is essential for repression of high energy demanding cell processes such as protein synthesis. The most abundant effect was the constitutively high phosphorylation of ribosomal protein S6 (RPS6) in the snrk1α1/α2 mutant. RPS6 is a major target of TOR signalling and its phosphorylation correlates with translation. Further evidence for an antagonistic SnRK1 and TOR crosstalk comparable to the animal system was demonstrated by the in vivo interaction of SnRK1α1 and RAPTOR1B in the cytosol and by phosphorylation of RAPTOR1B by SnRK1α1 in kinase assays. Moreover, changed levels of phosphorylation states of several chloroplastic proteins in the snrk1α1/α2 mutant indicated an unexpected link to regulation of photosynthesis, the main energy source in plants. PMID:27545962

  1. Quantitative phosphoproteomics reveals the role of the AMPK plant ortholog SnRK1 as a metabolic master regulator under energy deprivation.

    PubMed

    Nukarinen, Ella; Nägele, Thomas; Pedrotti, Lorenzo; Wurzinger, Bernhard; Mair, Andrea; Landgraf, Ramona; Börnke, Frederik; Hanson, Johannes; Teige, Markus; Baena-Gonzalez, Elena; Dröge-Laser, Wolfgang; Weckwerth, Wolfram

    2016-01-01

    Since years, research on SnRK1, the major cellular energy sensor in plants, has tried to define its role in energy signalling. However, these attempts were notoriously hampered by the lethality of a complete knockout of SnRK1. Therefore, we generated an inducible amiRNA::SnRK1α2 in a snrk1α1 knock out background (snrk1α1/α2) to abolish SnRK1 activity to understand major systemic functions of SnRK1 signalling under energy deprivation triggered by extended night treatment. We analysed the in vivo phosphoproteome, proteome and metabolome and found that activation of SnRK1 is essential for repression of high energy demanding cell processes such as protein synthesis. The most abundant effect was the constitutively high phosphorylation of ribosomal protein S6 (RPS6) in the snrk1α1/α2 mutant. RPS6 is a major target of TOR signalling and its phosphorylation correlates with translation. Further evidence for an antagonistic SnRK1 and TOR crosstalk comparable to the animal system was demonstrated by the in vivo interaction of SnRK1α1 and RAPTOR1B in the cytosol and by phosphorylation of RAPTOR1B by SnRK1α1 in kinase assays. Moreover, changed levels of phosphorylation states of several chloroplastic proteins in the snrk1α1/α2 mutant indicated an unexpected link to regulation of photosynthesis, the main energy source in plants. PMID:27545962

  2. Enrichment Strategies in Phosphoproteomics.

    PubMed

    Leitner, Alexander

    2016-01-01

    The comprehensive study of the phosphoproteome is heavily dependent on appropriate enrichment strategies that are most often, but not exclusively, carried out on the peptide level. In this chapter, I give an overview of the most widely used techniques. In addition to dedicated antibodies, phosphopeptides are enriched by their selective interaction with metals in the form of chelated metal ions or metal oxides. The negative charge of the phosphate group is also exploited in a variety of chromatographic fractionation methods that include different types of ion exchange chromatography, hydrophilic interaction chromatography (HILIC), and electrostatic repulsion HILIC (ERLIC) chromatography. Selected examples from the literature will demonstrate how a combination of these techniques with current high-performance mass spectrometry enables the identification of thousands of phosphorylation sites from various sample types. PMID:26584921

  3. Dissecting Analogical Leveling Quantitatively: The Case of the Innovative Potential Suffix in Tokyo Japanese.

    ERIC Educational Resources Information Center

    Matsuda, Kenjiro

    1993-01-01

    Analogical leveling in progress of a potential suffix in Tokyo Japanese is analyzed within a quantitative model. The phenomenon is shown to be controlled by five factors: sociological variable complex, verb stem length, verb conjugation pattern, the following inflectional form, and embeddedness of the clause containing the suffix. (Contains 70…

  4. Dissecting Quantitative Trait Loci for Boron Efficiency across Multiple Environments in Brassica napus

    PubMed Central

    Zhao, Zunkang; Wu, Likun; Nian, Fuzhao; Ding, Guangda; Shi, Taoxiong; Zhang, Didi; Shi, Lei; Xu, Fangsen; Meng, Jinling

    2012-01-01

    High yield is the most important goal in crop breeding, and boron (B) is an essential micronutrient for plants. However, B deficiency, leading to yield decreases, is an agricultural problem worldwide. Brassica napus is one of the most sensitive crops to B deficiency, and considerable genotypic variation exists among different cultivars in response to B deficiency. To dissect the genetic basis of tolerance to B deficiency in B. napus, we carried out QTL analysis for seed yield and yield-related traits under low and normal B conditions using the double haploid population (TNDH) by two-year and the BQDH population by three-year field trials. In total, 80 putative QTLs and 42 epistatic interactions for seed yield, plant height, branch number, pod number, seed number, seed weight and B efficiency coefficient (BEC) were identified under low and normal B conditions, singly explaining 4.15–23.16% and 0.53–14.38% of the phenotypic variation. An additive effect of putative QTLs was a more important controlling factor than the additive-additive effect of epistatic interactions. Four QTL-by-environment interactions and 7 interactions between epistatic interactions and the environment contributed to 1.27–4.95% and 1.17–3.68% of the phenotypic variation, respectively. The chromosome region on A2 of SYLB-A2 for seed yield under low B condition and BEC-A2 for BEC in the two populations was equivalent to the region of a reported major QTL, BE1. The B. napus homologous genes of Bra020592 and Bra020595 mapped to the A2 region and were speculated to be candidate genes for B efficiency. These findings reveal the complex genetic basis of B efficiency in B. napus. They provide a basis for the fine mapping and cloning of the B efficiency genes and for breeding B-efficient cultivars by marker-assisted selection (MAS). PMID:23028855

  5. Clinical and Technical Phosphoproteomic Research

    PubMed Central

    2011-01-01

    An encouraging approach for the diagnosis and effective therapy of immunological pathologies, which would include cancer, is the identification of proteins and phosphorylated proteins. Disease proteomics, in particular, is a potentially useful method for this purpose. A key role is played by protein phosphorylation in the regulation of normal immunology disorders and targets for several new cancer drugs and drug candidates are cancer cells and protein kinases. Protein phosphorylation is a highly dynamic process. The functioning of new drugs is of major importance as is the selection of those patients who would respond best to a specific treatment regime. In all major aspects of cellular life signalling networks are key elements which play a major role in inter- and intracellular communications. They are involved in diverse processes such as cell-cycle progression, cellular metabolism, cell-cell communication and appropriate response to the cellular environment. A whole range of networks that are involved in the regulation of cell development, differentiation, proliferation, apoptosis, and immunologic responses is contained in the latter. It is so necessary to understand and monitor kinase signalling pathways in order to understand many immunology pathologies. Enrichment of phosphorylated proteins or peptides from tissue or bodily fluid samples is required. The application of technologies such as immunoproteomic techniques, phosphoenrichments and mass spectrometry (MS) is crucial for the identification and quantification of protein phosphorylation sites in order to advance in clinical research. Pharmacodynamic readouts of disease states and cellular drug responses in tumour samples will be provided as the field develops. We aim to detail the current and most useful techniques with research examples to isolate and carry out clinical phosphoproteomic studies which may be helpful for immunology and cancer research. Different phosphopeptide enrichment and quantitative

  6. Quantitative dissection and stoichiometry determination of the human SET1/MLL histone methyltransferase complexes.

    PubMed

    van Nuland, Rick; Smits, Arne H; Pallaki, Paschalina; Jansen, Pascal W T C; Vermeulen, Michiel; Timmers, H T Marc

    2013-05-01

    Methylation of lysine 4 on histone H3 (H3K4) at promoters is tightly linked to transcriptional regulation in human cells. At least six different COMPASS-like multisubunit (SET1/MLL) complexes that contain methyltransferase activity for H3K4 have been described, but a comprehensive and quantitative analysis of these SET1/MLL complexes is lacking. We applied label-free quantitative mass spectrometry to determine the subunit composition and stoichiometry of the human SET1/MLL complexes. We identified both known and novel, unique and shared interactors and determined their distribution and stoichiometry over the different SET1/MLL complexes. In addition to being a core COMPASS subunit, the Dpy30 protein is a genuine subunit of the NURF chromatin remodeling complex. Furthermore, we identified the Bod1 protein as a discriminator between the SET1B and SET1A complexes, and we show that the H3K36me-interactor Psip1 preferentially binds to the MLL2 complex. Finally, absolute protein quantification in crude lysates mirrors many of the observed SET1/MLL complex stoichiometries. Our findings provide a molecular framework for understanding the diversity and abundance of the different SET1/MLL complexes, which together establish the H3K4 methylation landscape in human cells. PMID:23508102

  7. The interplay of light and oxygen in the reactive oxygen stress response of Chlamydomonas reinhardtii dissected by quantitative mass spectrometry.

    PubMed

    Barth, Johannes; Bergner, Sonja Verena; Jaeger, Daniel; Niehues, Anna; Schulze, Stefan; Scholz, Martin; Fufezan, Christian

    2014-04-01

    Light and oxygen are factors that are very much entangled in the reactive oxygen species (ROS) stress response network in plants, algae and cyanobacteria. The first obligatory step in understanding the ROS network is to separate these responses. In this study, a LC-MS/MS based quantitative proteomic approach was used to dissect the responses of Chlamydomonas reinhardtii to ROS, light and oxygen employing an interlinked experimental setup. Application of novel bioinformatics tools allow high quality retention time alignment to be performed on all LC-MS/MS runs increasing confidence in protein quantification, overall sequence coverage and coverage of all treatments measured. Finally advanced hierarchical clustering yielded 30 communities of co-regulated proteins permitting separation of ROS related effects from pure light effects (induction and repression). A community termed redox(II) was identified that shows additive effects of light and oxygen with light as the first obligatory step. Another community termed 4-down was identified that shows repression as an effect of light but only in the absence of oxygen indicating ROS regulation, for example, possibly via product feedback inhibition because no ROS damage is occurring. In summary the data demonstrate the importance of separating light, O₂ and ROS responses to define marker genes for ROS responses. As revealed in this study, an excellent candidate is DHAR with strong ROS dependent induction profiles.

  8. Phosphoproteome analysis of formalin-fixed and paraffin-embedded tissue sections mounted on microscope slides.

    PubMed

    Wakabayashi, Masaki; Yoshihara, Hiroki; Masuda, Takeshi; Tsukahara, Mai; Sugiyama, Naoyuki; Ishihama, Yasushi

    2014-02-01

    Formalin-fixed and paraffin-embedded (FFPE) sections mounted on microscope slides are one of the largest available resources for retrospective research on various diseases, but quantitative phosphoproteome analysis of FFPE sections has never been achieved because of the extreme difficulty of procuring sufficient phosphopeptides from the limited amounts of proteins on the slides. Here, we present the first protocol for quantitative phosphoproteome analysis of FFPE sections by utilizing phase-transfer surfactant-aided extraction/tryptic digestion of FFPE proteins followed by high-recovery phosphopeptide enrichment via lactic acid-modified titania chromatography. We established that FFPE sections retain a similar phosphoproteome to fresh tissue specimens during storage for at least 9 months, confirming the utility of our method for evaluating phosphorylation profiles in various diseases. We also verified that chemical labeling based on reductive dimethylation of amino groups was feasible for quantitative phosphoproteome analysis of FFPE samples on slides. Furthermore, we improved the LC-MS sensitivity by miniaturizing nanoLC columns to 25 μm inner diameter. With this system, we could identify 1090 phosphopeptides from a single FFPE section obtained from a microscope slide, containing 25.2 ± 5.4 μg of proteins. This protocol should be useful for large-scale phosphoproteome analysis of archival FFPE slides, especially scarce samples from patients with rare diseases.

  9. Quantitative trait loci mapping and genetic dissection for lint percentage in upland cotton (Gossypium hirsutum).

    PubMed

    Wang, Min; Li, Chengqi; Wang, Qinglian

    2014-08-01

    Lint percentage is an important character of cotton yield components and it is also correlated with cotton fibre development. In this study, we used a high lint percentage variety, Baimian1, and a low lint percentage, TM-1 genetic standard for Gossypium hirsutum, as parents to construct a mapping populations in upland cotton (G. hirsutum). A quantitative trait locus/loci (QTL) analysis of lint percentage was performed by using two mapping procedures; composite interval mapping (CIM), inclusive composite interval mapping (ICIM) and the F2:3 populations in 2 years. Six main-effect QTL (M-QTL) for lint percentage (four significant and two suggestive) were detected in both years by CIM, and were located on chr. 3, chr. 19, chr. 26 and chr. 5/chr. 19. Of the six QTL, marker intervals and favourable gene sources of the significant M-QTL, qLP-3(2010) and qLP-3(2011) were consistent. These QTL were also detected by ICIM, and therefore, should preferentially be used for markerassisted selection (MAS) of lint percentage. Another M-QTL, qLP-19(2010), was detected by two mapping procedures, and it could also be a candidate for MAS. We detected the interaction between two M-QTL and environment, and 11 epistatic QTL (E-QTL) and their interaction with environment by using ICIM. The study also found two EST-SSRs, NAU1187 and NAU1255, linked to M-QTL for lint percentage that could be candidate markers affecting cotton fibre development.

  10. Quantitative trait loci mapping and genetic dissection for lint percentage in upland cotton (Gossypium hirsutum).

    PubMed

    Wang, Min; Li, Chengqi; Wang, Qinglian

    2014-08-01

    Lint percentage is an important character of cotton yield components and it is also correlated with cotton fibre development. In this study, we used a high lint percentage variety, Baimian1, and a low lint percentage, TM-1 genetic standard for Gossypium hirsutum, as parents to construct a mapping populations in upland cotton (G. hirsutum). A quantitative trait locus/loci (QTL) analysis of lint percentage was performed by using two mapping procedures; composite interval mapping (CIM), inclusive composite interval mapping (ICIM) and the F2:3 populations in 2 years. Six main-effect QTL (M-QTL) for lint percentage (four significant and two suggestive) were detected in both years by CIM, and were located on chr. 3, chr. 19, chr. 26 and chr. 5/chr. 19. Of the six QTL, marker intervals and favourable gene sources of the significant M-QTL, qLP-3(2010) and qLP-3(2011) were consistent. These QTL were also detected by ICIM, and therefore, should preferentially be used for markerassisted selection (MAS) of lint percentage. Another M-QTL, qLP-19(2010), was detected by two mapping procedures, and it could also be a candidate for MAS. We detected the interaction between two M-QTL and environment, and 11 epistatic QTL (E-QTL) and their interaction with environment by using ICIM. The study also found two EST-SSRs, NAU1187 and NAU1255, linked to M-QTL for lint percentage that could be candidate markers affecting cotton fibre development. PMID:25189232

  11. Genetic dissection of fruiting body-related traits using quantitative trait loci mapping in Lentinula edodes.

    PubMed

    Gong, Wen-Bing; Li, Lei; Zhou, Yan; Bian, Yin-Bing; Kwan, Hoi-Shan; Cheung, Man-Kit; Xiao, Yang

    2016-06-01

    To provide a better understanding of the genetic architecture of fruiting body formation of Lentinula edodes, quantitative trait loci (QTLs) mapping was employed to uncover the loci underlying seven fruiting body-related traits (FBRTs). An improved L. edodes genetic linkage map, comprising 572 markers on 12 linkage groups with a total map length of 983.7 cM, was constructed by integrating 82 genomic sequence-based insertion-deletion (InDel) markers into a previously published map. We then detected a total of 62 QTLs for seven target traits across two segregating testcross populations, with individual QTLs contributing 5.5 %-30.2 % of the phenotypic variation. Fifty-three out of the 62 QTLs were clustered in six QTL hotspots, suggesting the existence of main genomic regions regulating the morphological characteristics of fruiting bodies in L. edodes. A stable QTL hotspot on MLG2, containing QTLs for all investigated traits, was identified in both testcross populations. QTLs for related traits were frequently co-located on the linkage groups, demonstrating the genetic basis for phenotypic correlation of traits. Meta-QTL (mQTL) analysis was performed and identified 16 mQTLs with refined positions and narrow confidence intervals (CIs). Nine genes, including those encoding MAP kinase, blue-light photoreceptor, riboflavin-aldehyde-forming enzyme and cyclopropane-fatty-acyl-phospholipid synthase, and cytochrome P450s, were likely to be candidate genes controlling the shape of fruiting bodies. The study has improved our understanding of the genetic architecture of fruiting body formation in L. edodes. To our knowledge, this is the first genome-wide QTL detection of FBRTs in L. edodes. The improved genetic map, InDel markers and QTL hotspot regions revealed here will assist considerably in the conduct of future genetic and breeding studies of L. edodes.

  12. Dissecting the Paclitaxel-Microtubule Association: Quantitative Assessment of the 2′-OH Group†

    PubMed Central

    Sharma, Shubhada; Lagisetti, Chandraiah; Poliks, Barbara; Coates, Robert M.; Kingston, David G. I.; Bane, Susan

    2013-01-01

    Paclitaxel (PTX) is a microtubule-stabilizing agent that is widely used in cancer chemotherapy. This structurally complex natural product acts by binding to β-tubulin in assembled microtubules. The 2′-hydroxyl group in the flexible side chain of PTX is an absolute requirement for activity, but its precise role in the drug-receptor interaction has not been specifically investigated. The contribution of the 2′-OH group to the affinity and tubulin-assembly efficacy of PTX has been evaluated through quantitative analysis of PTX derivatives possessing side chain deletions: 2′-deoxy-PTX, N-debenzoyl-2′-deoxy-PTX and baccatin III. The affinity of 2′-deoxy-PTX for stabilized microtubules was more than 100-fold less than that of PTX and only about 3-fold greater than the microtubule affinity of baccatin III. No microtubule binding activity was detected for the analog N-debenzoyl-2′-deoxy-PTX. The tubulin-assembly efficacy of each ligand was in concordance with the microtubule binding affinity, as was the trend in cytotoxicities. Molecular dynamics simulations revealed that the 2′-OH group of PTX can form a persistent hydrogen bond with D26 within the microtubule binding site. The absence of this interaction between 2′-deoxy-PTX and the receptor can account for the difference in binding free energy. Computational analyses also provide a possible explanation for why N-debenzoyl-2′-deoxy-PTX is inactive, in spite of the fact that it is essentially a substituted baccatin III. We propose that the hydrogen bonding interaction between the 2′-OH group and D26 is the most important stabilizing interaction that PTX forms with tubulin in the region of the C-13 side chain. We further hypothesize that the substituents at the 3′-position function to orient the 2′-OH group for a productive hydrogen bonding interaction with the protein. PMID:23473345

  13. Combining Quantitative Genetics Approaches with Regulatory Network Analysis to Dissect the Complex Metabolism of the Maize Kernel1[OPEN

    PubMed Central

    Wen, Weiwei; Liu, Haijun; Yang, Ning; Luo, Jie; Xiao, Yingjie; Pan, Qingchun; Tohge, Takayuki; Fernie, Alisdair R.; Yan, Jianbing

    2016-01-01

    Metabolic quantitative trait locus (QTL) studies have allowed us to better understand the genetic architecture underlying naturally occurring plant metabolic variance. Here, we use two recombinant inbred line (RIL) populations to dissect the genetic architecture of natural variation of 155 metabolites measured in the mature maize (Zea mays) kernel. Overall, linkage mapping identified 882 metabolic QTLs in both RIL populations across two environments, with an average of 2.1 QTLs per metabolite. A large number of metabolic QTLs (more than 65%) were identified with moderate effects (r2 = 2.1%–10%), while a small portion (less than 35%) showed major effects (r2 > 10%). Epistatic interactions between these identified loci were detected for more than 30% of metabolites (with the proportion of phenotypic variance ranging from 1.6% to 37.8%), implying that genetic epistasis is not negligible in determining metabolic variation. In total, 57 QTLs were validated by our previous genome-wide association study on the same metabolites that provided clues for exploring the underlying genes. A gene regulatory network associated with the flavonoid metabolic pathway was constructed based on the transcriptional variations of 28,769 genes in kernels (15 d after pollination) of 368 maize inbred lines. A large number of genes (34 of 58) in this network overlapped with previously defined genes controlled by maize PERICARP COLOR1, while three of them were identified here within QTL intervals for multiple flavonoids. The deeply characterized RIL populations, elucidation of metabolic phenotypes, and identification of candidate genes lay the foundation for maize quality improvement. PMID:26556794

  14. Phosphoproteomics and Lung Cancer Research

    PubMed Central

    López, Elena; Cho, William C. S.

    2012-01-01

    Massive evidence suggests that genetic abnormalities contribute to the development of lung cancer. These molecular abnormalities may serve as diagnostic, prognostic and predictive biomarkers for this deadly disease. It is imperative to search these biomarkers in different tumorigenesis pathways so as to provide the most appropriate therapy for each individual patient with lung malignancy. Phosphoproteomics is a promising technology for the identification of biomarkers and novel therapeutic targets for cancer. Thousands of proteins interact via physical and chemical association. Moreover, some proteins can covalently modify other proteins post-translationally. These post-translational modifications ultimately give rise to the emergent functions of cells in sequence, space and time. Phosphoproteomics clinical researches imply the comprehensive analysis of the proteins that are expressed in cells or tissues and can be employed at different stages. In addition, understanding the functions of phosphorylated proteins requires the study of proteomes as linked systems rather than collections of individual protein molecules. In fact, proteomics approaches coupled with affinity chromatography strategies followed by mass spectrometry have been used to elucidate relevant biological questions. This article will discuss the relevant clues of post-translational modifications, phosphorylated proteins, and useful proteomics approaches to identify molecular cancer signatures. The recent progress in phosphoproteomics research in lung cancer will be also discussed. PMID:23202899

  15. Influence of sample preparation and reliability of automated numerical refocusing in stain-free analysis of dissected tissues with quantitative phase digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Kemper, Björn; Lenz, Philipp; Bettenworth, Dominik; Krausewitz, Philipp; Domagk, Dirk; Ketelhut, Steffi

    2015-05-01

    Digital holographic microscopy (DHM) has been demonstrated to be a versatile tool for high resolution non-destructive quantitative phase imaging of surfaces and multi-modal minimally-invasive monitoring of living cell cultures in-vitro. DHM provides quantitative monitoring of physiological processes through functional imaging and structural analysis which, for example, gives new insight into signalling of cellular water permeability and cell morphology changes due to toxins and infections. Also the analysis of dissected tissues quantitative DHM phase contrast prospects application fields by stain-free imaging and the quantification of tissue density changes. We show that DHM allows imaging of different tissue layers with high contrast in unstained tissue sections. As the investigation of fixed samples represents a very important application field in pathology, we also analyzed the influence of the sample preparation. The retrieved data demonstrate that the quality of quantitative DHM phase images of dissected tissues depends strongly on the fixing method and common staining agents. As in DHM the reconstruction is performed numerically, multi-focus imaging is achieved from a single digital hologram. Thus, we evaluated the automated refocussing feature of DHM for application on different types of dissected tissues and revealed that on moderately stained samples highly reproducible holographic autofocussing can be achieved. Finally, it is demonstrated that alterations of the spatial refractive index distribution in murine and human tissue samples represent a reliable absolute parameter that is related of different degrees of inflammation in experimental colitis and Crohn's disease. This paves the way towards the usage of DHM in digital pathology for automated histological examinations and further studies to elucidate the translational potential of quantitative phase microscopy for the clinical management of patients, e.g., with inflammatory bowel disease.

  16. Dissecting the Dissection

    PubMed Central

    Sherif, Hisham M.F.

    2015-01-01

    Aortic dissection remains one of the most devastating diseases. Current practice guidelines provide diagnostic and therapeutic interventions based primarily on the aortic diameter. The level of evidence supporting these recommendations is Level C or “Expert Opinion” Since aortic dissection is a catastrophic structural failure, its investigation along the guidelines of accident investigation may offer a useful alternative, utilizing process mapping and root-cause analysis methodology. Since the objective of practice guidelines is to address the risk of serious events, on the utilization of a probabilistic predictive modeling methodology, using bioinformatics tools, may offer a more comprehensive risk assessment. PMID:27069940

  17. Sperm phosphoproteomics: historical perspectives and current methodologies

    PubMed Central

    Porambo, James R; Salicioni, Ana M; Visconti, Pablo E; Platt, Mark D

    2013-01-01

    Mammalian sperm are differentiated germ cells that transfer genetic material from the male to the female. Owing to this essential role in the reproductive process, an understanding of the complex mechanisms that underlie sperm function has implications ranging from the development of novel contraceptives to the treatment of male infertility. While the importance of phosphorylation in sperm differentiation, maturation and fertilization has been well established, the ability to directly determine the sites of phosphorylation within sperm proteins and to quantitate the extent of phosphorylation at these sites is a recent development that has relied almost exclusively on advances in the field of proteomics. This review will summarize the work that has been carried out to date on sperm phosphoproteomics and discuss how the resulting qualitative and quantitative information has been used to provide insight into the manner in which protein phosphorylation events modulate sperm function. The authors also present the proteomics process as it is most often utilized for the elucidation of protein expression, with a particular emphasis on the way in which the process has been modified for the analysis of protein phosphorylation in sperm. PMID:23194270

  18. Phosphoproteomics for oncology discovery and treatment.

    PubMed

    Stern, David F

    2005-08-01

    Reversible protein phosphorylation is one of the most important forms of cellular regulation. For this reason, phosphoproteomic analysis of protein phosphorylation in cells is a powerful tool for evaluating the functional status of cells. The importance of protein kinase-regulated signal transduction pathways in human cancer has led to the development of drugs that inhibit protein kinases at the apex or intermediary levels of these pathways. Phosphoproteomic analysis of these signalling pathways will provide important insights into the operation and connectivity of these pathways that will facilitate the identification of the best targets for cancer therapies. Moreover, phosphoproteomic analysis of individual tumours will help match targeted cancer drugs to the appropriate patients.

  19. Comparative phosphoproteomics of zebrafish Fyn/Yes morpholino knockdown embryos.

    PubMed

    Lemeer, Simone; Jopling, Chris; Gouw, Joost; Mohammed, Shabaz; Heck, Albert J R; Slijper, Monique; den Hertog, Jeroen

    2008-11-01

    The coordinated movement of cells is indispensable for normal vertebrate gastrulation. Several important players and signaling pathways have been identified in convergence and extension (CE) cell movements during gastrulation, including non-canonical Wnt signaling. Fyn and Yes, members of the Src family of kinases, are key regulators of CE movements as well. Here we investigated signaling pathways in early development by comparison of the phosphoproteome of wild type zebrafish embryos with Fyn/Yes knockdown embryos that display specific CE cell movement defects. For quantitation we used differential stable isotope labeling by reductive amination of peptides. Equal amounts of labeled peptides from wild type and Fyn/Yes knockdown embryos were mixed and analyzed by on-line reversed phase TiO(2)-reversed phase LC-MS/MS. Phosphorylated and non-phosphorylated peptides were quantified, and significant changes in protein expression and/or phosphorylation were detected. We identified 348 phosphoproteins of which 69 showed a decrease in phosphorylation in Fyn/Yes knockdown embryos and 72 showed an increase in phosphorylation. Among these phosphoproteins were known regulators of cell movements, including Adducin and PDLIM5. Our results indicate that quantitative phosphoproteomics combined with morpholino-mediated knockdowns can be used to identify novel signaling pathways that act in zebrafish development in vivo.

  20. Phosphoproteomic Analyses Reveal Signaling Pathways That Facilitate Lytic Gammaherpesvirus Replication

    PubMed Central

    Stahl, James A.; Chavan, Shweta S.; Sifford, Jeffrey M.; MacLeod, Veronica; Voth, Daniel E.; Edmondson, Ricky D.; Forrest, J. Craig

    2013-01-01

    Lytic gammaherpesvirus (GHV) replication facilitates the establishment of lifelong latent infection, which places the infected host at risk for numerous cancers. As obligate intracellular parasites, GHVs must control and usurp cellular signaling pathways in order to successfully replicate, disseminate to stable latency reservoirs in the host, and prevent immune-mediated clearance. To facilitate a systems-level understanding of phosphorylation-dependent signaling events directed by GHVs during lytic replication, we utilized label-free quantitative mass spectrometry to interrogate the lytic replication cycle of murine gammaherpesvirus-68 (MHV68). Compared to controls, MHV68 infection regulated by 2-fold or greater ca. 86% of identified phosphopeptides – a regulatory scale not previously observed in phosphoproteomic evaluations of discrete signal-inducing stimuli. Network analyses demonstrated that the infection-associated induction or repression of specific cellular proteins globally altered the flow of information through the host phosphoprotein network, yielding major changes to functional protein clusters and ontologically associated proteins. A series of orthogonal bioinformatics analyses revealed that MAPK and CDK-related signaling events were overrepresented in the infection-associated phosphoproteome and identified 155 host proteins, such as the transcription factor c-Jun, as putative downstream targets. Importantly, functional tests of bioinformatics-based predictions confirmed ERK1/2 and CDK1/2 as kinases that facilitate MHV68 replication and also demonstrated the importance of c-Jun. Finally, a transposon-mutant virus screen identified the MHV68 cyclin D ortholog as a viral protein that contributes to the prominent MAPK/CDK signature of the infection-associated phosphoproteome. Together, these analyses enhance an understanding of how GHVs reorganize and usurp intracellular signaling networks to facilitate infection and replication. PMID:24068923

  1. Phosphoproteomic analyses reveal signaling pathways that facilitate lytic gammaherpesvirus replication.

    PubMed

    Stahl, James A; Chavan, Shweta S; Sifford, Jeffrey M; MacLeod, Veronica; Voth, Daniel E; Edmondson, Ricky D; Forrest, J Craig

    2013-09-01

    Lytic gammaherpesvirus (GHV) replication facilitates the establishment of lifelong latent infection, which places the infected host at risk for numerous cancers. As obligate intracellular parasites, GHVs must control and usurp cellular signaling pathways in order to successfully replicate, disseminate to stable latency reservoirs in the host, and prevent immune-mediated clearance. To facilitate a systems-level understanding of phosphorylation-dependent signaling events directed by GHVs during lytic replication, we utilized label-free quantitative mass spectrometry to interrogate the lytic replication cycle of murine gammaherpesvirus-68 (MHV68). Compared to controls, MHV68 infection regulated by 2-fold or greater ca. 86% of identified phosphopeptides - a regulatory scale not previously observed in phosphoproteomic evaluations of discrete signal-inducing stimuli. Network analyses demonstrated that the infection-associated induction or repression of specific cellular proteins globally altered the flow of information through the host phosphoprotein network, yielding major changes to functional protein clusters and ontologically associated proteins. A series of orthogonal bioinformatics analyses revealed that MAPK and CDK-related signaling events were overrepresented in the infection-associated phosphoproteome and identified 155 host proteins, such as the transcription factor c-Jun, as putative downstream targets. Importantly, functional tests of bioinformatics-based predictions confirmed ERK1/2 and CDK1/2 as kinases that facilitate MHV68 replication and also demonstrated the importance of c-Jun. Finally, a transposon-mutant virus screen identified the MHV68 cyclin D ortholog as a viral protein that contributes to the prominent MAPK/CDK signature of the infection-associated phosphoproteome. Together, these analyses enhance an understanding of how GHVs reorganize and usurp intracellular signaling networks to facilitate infection and replication.

  2. Refined phosphopeptide enrichment by phosphate additive and the analysis of human brain phosphoproteome.

    PubMed

    Tan, Haiyan; Wu, Zhiping; Wang, Hong; Bai, Bing; Li, Yuxin; Wang, Xusheng; Zhai, Bo; Beach, Thomas G; Peng, Junmin

    2015-01-01

    Alzheimer's disease (AD) is the most common form of dementia, characterized by progressive loss of cognitive function. One of the pathological hallmarks of AD is the formation of neurofibrillary tangles composed of abnormally hyperphosphorylated tau protein, but global deregulation of protein phosphorylation in AD is not well analyzed. Here, we report a pilot investigation of AD phosphoproteome by titanium dioxide enrichment coupled with high resolution LC-MS/MS. During the optimization of the enrichment method, we found that phosphate ion at a low concentration (e.g. 1 mM) worked efficiently as a nonphosphopeptide competitor to reduce background. The procedure was further tuned with respect to peptide-to-bead ratio, phosphopeptide recovery, and purity. Using this refined method and 9 h LC-MS/MS, we analyzed phosphoproteome in one milligram of digested AD brain lysate, identifying 5243 phosphopeptides containing 3715 nonredundant phosphosites on 1455 proteins, including 31 phosphosites on the tau protein. This modified enrichment method is simple and highly efficient. The AD case study demonstrates its feasibility of dissecting phosphoproteome in a limited amount of postmortem human brain. All MS data have been deposited in the ProteomeXchange with identifier PXD001180 (http://proteomecentral.proteomexchange.org/dataset/PXD001180). PMID:25307156

  3. Technical phosphoproteomic and bioinformatic tools useful in cancer research

    PubMed Central

    2011-01-01

    Reversible protein phosphorylation is one of the most important forms of cellular regulation. Thus, phosphoproteomic analysis of protein phosphorylation in cells is a powerful tool to evaluate cell functional status. The importance of protein kinase-regulated signal transduction pathways in human cancer has led to the development of drugs that inhibit protein kinases at the apex or intermediary levels of these pathways. Phosphoproteomic analysis of these signalling pathways will provide important insights for operation and connectivity of these pathways to facilitate identification of the best targets for cancer therapies. Enrichment of phosphorylated proteins or peptides from tissue or bodily fluid samples is required. The application of technologies such as phosphoenrichments, mass spectrometry (MS) coupled to bioinformatics tools is crucial for the identification and quantification of protein phosphorylation sites for advancing in such relevant clinical research. A combination of different phosphopeptide enrichments, quantitative techniques and bioinformatic tools is necessary to achieve good phospho-regulation data and good structural analysis of protein studies. The current and most useful proteomics and bioinformatics techniques will be explained with research examples. Our aim in this article is to be helpful for cancer research via detailing proteomics and bioinformatic tools. PMID:21967744

  4. Technical phosphoproteomic and bioinformatic tools useful in cancer research.

    PubMed

    López, Elena; Wesselink, Jan-Jaap; López, Isabel; Mendieta, Jesús; Gómez-Puertas, Paulino; Muñoz, Sarbelio Rodríguez

    2011-01-01

    Reversible protein phosphorylation is one of the most important forms of cellular regulation. Thus, phosphoproteomic analysis of protein phosphorylation in cells is a powerful tool to evaluate cell functional status. The importance of protein kinase-regulated signal transduction pathways in human cancer has led to the development of drugs that inhibit protein kinases at the apex or intermediary levels of these pathways. Phosphoproteomic analysis of these signalling pathways will provide important insights for operation and connectivity of these pathways to facilitate identification of the best targets for cancer therapies. Enrichment of phosphorylated proteins or peptides from tissue or bodily fluid samples is required. The application of technologies such as phosphoenrichments, mass spectrometry (MS) coupled to bioinformatics tools is crucial for the identification and quantification of protein phosphorylation sites for advancing in such relevant clinical research. A combination of different phosphopeptide enrichments, quantitative techniques and bioinformatic tools is necessary to achieve good phospho-regulation data and good structural analysis of protein studies. The current and most useful proteomics and bioinformatics techniques will be explained with research examples. Our aim in this article is to be helpful for cancer research via detailing proteomics and bioinformatic tools. PMID:21967744

  5. Debating Dissection.

    ERIC Educational Resources Information Center

    Orlans, F. Barbara

    1988-01-01

    Argues the pros, cons, and alternatives to animal dissection. Discusses several cases involving student objections and the enactment of a California state law which upholds the right of a student to conscientious objection to dissection involving the harmful or destructive use of animals. (RT)

  6. Phosphoproteomic Analysis of Cell-Based Resistance to BRAF Inhibitor Therapy in Melanoma

    PubMed Central

    Parker, Robert; Vella, Laura J.; Xavier, Dylan; Amirkhani, Ardeshir; Parker, Jimmy; Cebon, Jonathan; Molloy, Mark P.

    2015-01-01

    The treatment of melanoma by targeted inhibition of the mutated kinase BRAF with small molecules only temporarily suppresses metastatic disease. In the face of chemical inhibition tumor plasticity, both innate and adaptive, promotes survival through the biochemical and genetic reconfiguration of cellular pathways that can engage proliferative and migratory systems. To investigate this process, high-resolution mass spectrometry was used to characterize the phosphoproteome of this transition in vitro. A simple and accurate, label-free quantitative method was used to localize and quantitate thousands of phosphorylation events. We also correlated changes in the phosphoproteome with the proteome to more accurately determine changes in the activity of regulatory kinases determined by kinase landscape profiling. The abundance of phosphopeptides with sites that function in cytoskeletal regulation, GTP/GDP exchange, protein kinase C, IGF signaling, and melanosome maturation were highly divergent after transition to a drug resistant phenotype. PMID:26029660

  7. Phosphoproteomics analysis of a clinical Mycobacterium tuberculosis Beijing isolate: expanding the mycobacterial phosphoproteome catalog

    PubMed Central

    Fortuin, Suereta; Tomazella, Gisele G.; Nagaraj, Nagarjuna; Sampson, Samantha L.; Gey van Pittius, Nicolaas C.; Soares, Nelson C.; Wiker, Harald G.; de Souza, Gustavo A.; Warren, Robin M.

    2015-01-01

    Reversible protein phosphorylation, regulated by protein kinases and phosphatases, mediates a switch between protein activity and cellular pathways that contribute to a large number of cellular processes. The Mycobacterium tuberculosis genome encodes 11 Serine/Threonine kinases (STPKs) which show close homology to eukaryotic kinases. This study aimed to elucidate the phosphoproteomic landscape of a clinical isolate of M. tuberculosis. We performed a high throughput mass spectrometric analysis of proteins extracted from an early-logarithmic phase culture. Whole cell lysate proteins were processed using the filter-aided sample preparation method, followed by phosphopeptide enrichment of tryptic peptides by strong cation exchange (SCX) and Titanium dioxide (TiO2) chromatography. The MaxQuant quantitative proteomics software package was used for protein identification. Our analysis identified 414 serine/threonine/tyrosine phosphorylated sites, with a distribution of S/T/Y sites; 38% on serine, 59% on threonine and 3% on tyrosine; present on 303 unique peptides mapping to 214 M. tuberculosis proteins. Only 45 of the S/T/Y phosphorylated proteins identified in our study had been previously described in the laboratory strain H37Rv, confirming previous reports. The remaining 169 phosphorylated proteins were newly identified in this clinical M. tuberculosis Beijing strain. We identified 5 novel tyrosine phosphorylated proteins. These findings not only expand upon our current understanding of the protein phosphorylation network in clinical M. tuberculosis but the data set also further extends and complements previous knowledge regarding phosphorylated peptides and phosphorylation sites in M. tuberculosis. PMID:25713560

  8. Phosphoproteomics analysis of a clinical Mycobacterium tuberculosis Beijing isolate: expanding the mycobacterial phosphoproteome catalog.

    PubMed

    Fortuin, Suereta; Tomazella, Gisele G; Nagaraj, Nagarjuna; Sampson, Samantha L; Gey van Pittius, Nicolaas C; Soares, Nelson C; Wiker, Harald G; de Souza, Gustavo A; Warren, Robin M

    2015-01-01

    Reversible protein phosphorylation, regulated by protein kinases and phosphatases, mediates a switch between protein activity and cellular pathways that contribute to a large number of cellular processes. The Mycobacterium tuberculosis genome encodes 11 Serine/Threonine kinases (STPKs) which show close homology to eukaryotic kinases. This study aimed to elucidate the phosphoproteomic landscape of a clinical isolate of M. tuberculosis. We performed a high throughput mass spectrometric analysis of proteins extracted from an early-logarithmic phase culture. Whole cell lysate proteins were processed using the filter-aided sample preparation method, followed by phosphopeptide enrichment of tryptic peptides by strong cation exchange (SCX) and Titanium dioxide (TiO2) chromatography. The MaxQuant quantitative proteomics software package was used for protein identification. Our analysis identified 414 serine/threonine/tyrosine phosphorylated sites, with a distribution of S/T/Y sites; 38% on serine, 59% on threonine and 3% on tyrosine; present on 303 unique peptides mapping to 214 M. tuberculosis proteins. Only 45 of the S/T/Y phosphorylated proteins identified in our study had been previously described in the laboratory strain H37Rv, confirming previous reports. The remaining 169 phosphorylated proteins were newly identified in this clinical M. tuberculosis Beijing strain. We identified 5 novel tyrosine phosphorylated proteins. These findings not only expand upon our current understanding of the protein phosphorylation network in clinical M. tuberculosis but the data set also further extends and complements previous knowledge regarding phosphorylated peptides and phosphorylation sites in M. tuberculosis.

  9. Genome-wide conserved non-coding microsatellite (CNMS) marker-based integrative genetical genomics for quantitative dissection of seed weight in chickpea.

    PubMed

    Bajaj, Deepak; Saxena, Maneesha S; Kujur, Alice; Das, Shouvik; Badoni, Saurabh; Tripathi, Shailesh; Upadhyaya, Hari D; Gowda, C L L; Sharma, Shivali; Singh, Sube; Tyagi, Akhilesh K; Parida, Swarup K

    2015-03-01

    Phylogenetic footprinting identified 666 genome-wide paralogous and orthologous CNMS (conserved non-coding microsatellite) markers from 5'-untranslated and regulatory regions (URRs) of 603 protein-coding chickpea genes. The (CT)n and (GA)n CNMS carrying CTRMCAMV35S and GAGA8BKN3 regulatory elements, respectively, are abundant in the chickpea genome. The mapped genic CNMS markers with robust amplification efficiencies (94.7%) detected higher intraspecific polymorphic potential (37.6%) among genotypes, implying their immense utility in chickpea breeding and genetic analyses. Seventeen differentially expressed CNMS marker-associated genes showing strong preferential and seed tissue/developmental stage-specific expression in contrasting genotypes were selected to narrow down the gene targets underlying seed weight quantitative trait loci (QTLs)/eQTLs (expression QTLs) through integrative genetical genomics. The integration of transcript profiling with seed weight QTL/eQTL mapping, molecular haplotyping, and association analyses identified potential molecular tags (GAGA8BKN3 and RAV1AAT regulatory elements and alleles/haplotypes) in the LOB-domain-containing protein- and KANADI protein-encoding transcription factor genes controlling the cis-regulated expression for seed weight in the chickpea. This emphasizes the potential of CNMS marker-based integrative genetical genomics for the quantitative genetic dissection of complex seed weight in chickpea.

  10. Aortic dissection.

    PubMed

    Nienaber, Christoph A; Clough, Rachel E; Sakalihasan, Natzi; Suzuki, Toru; Gibbs, Richard; Mussa, Firas; Jenkins, Michael T; Thompson, Matt M; Evangelista, Arturo; Yeh, James S M; Cheshire, Nicholas; Rosendahl, Ulrich; Pepper, John

    2016-01-01

    Aortic dissection is a life-threatening condition caused by a tear in the intimal layer of the aorta or bleeding within the aortic wall, resulting in the separation (dissection) of the layers of the aortic wall. Aortic dissection is most common in those 65-75 years of age, with an incidence of 35 cases per 100,000 people per year in this population. Other risk factors include hypertension, dyslipidaemia and genetic disorders that involve the connective tissue, such as Marfan syndrome. Swift diagnostic confirmation and adequate treatment are crucial in managing affected patients. Contemporary management is multidisciplinary and includes serial non-invasive imaging, biomarker testing and genetic risk profiling for aortopathy. The choice of approach for repairing or replacing the damaged region of the aorta depends on the severity and the location of the dissection and the risks of complication from surgery. Open surgical repair is most commonly used for dissections involving the ascending aorta and the aortic arch, whereas minimally invasive endovascular intervention is appropriate for descending aorta dissections that are complicated by rupture, malperfusion, ongoing pain, hypotension or imaging features of high risk. Recent advances in the understanding of the underlying pathophysiology of aortic dissection have led to more patients being considered at substantial risk of complications and, therefore, in need of endovascular intervention rather than only medical or surgical intervention. PMID:27440162

  11. Dissecting quantitative trait variation in the resequencing era: complementarity of bi-parental, multi-parental and association panels.

    PubMed

    Pascual, Laura; Albert, Elise; Sauvage, Christopher; Duangjit, Janejira; Bouchet, Jean-Paul; Bitton, Frédérique; Desplat, Nelly; Brunel, Dominique; Le Paslier, Marie-Christine; Ranc, Nicolas; Bruguier, Laure; Chauchard, Betty; Verschave, Philippe; Causse, Mathilde

    2016-01-01

    Quantitative trait loci (QTL) have been identified using traditional linkage mapping and positional cloning identified several QTLs. However linkage mapping is limited to the analysis of traits differing between two lines and the impact of the genetic background on QTL effect has been underlined. Genome-wide association studies (GWAs) were proposed to circumvent these limitations. In tomato, we have shown that GWAs is possible, using the admixed nature of cherry tomato genomes that reduces the impact of population structure. Nevertheless, GWAs success might be limited due to the low decay of linkage disequilibrium, which varies along the genome in this species. Multi-parent advanced generation intercross (MAGIC) populations offer an alternative to traditional linkage and GWAs by increasing the precision of QTL mapping. We have developed a MAGIC population by crossing eight tomato lines whose genomes were resequenced. We showed the potential of the MAGIC population when coupled with whole genome sequencing to detect candidate single nucleotide polymorphisms (SNPs) underlying the QTLs. QTLs for fruit quality traits were mapped and related to the variations detected at the genome sequence and expression levels. The advantages and limitations of the three types of population, in the context of the available genome sequence and resequencing facilities, are discussed. PMID:26566830

  12. Applicability of a "speed" congenic strategy to dissect blood pressure quantitative trait loci on rat chromosome 2.

    PubMed

    Jeffs, B; Negrin, C D; Graham, D; Clark, J S; Anderson, N H; Gauguier, D; Dominiczak, A F

    2000-01-01

    The identification of any quantitative trait locus (QTL) via a genome scan is only the first step toward the ultimate goal of gene identification. The next step is the production of congenic strains by which the existence of a QTL may be verified and the implicated chromosomal region be reduced to a size applicable to positional cloning of the causal gene. We used a speed congenic breeding protocol previously verified in mice for 2 blood pressure QTLs on rat chromosome 2. Four congenic strains were produced through introgression of various segments of chromosome 2 from Wistar-Kyoto rats from Glasgow colonies [WKY((Gla)) rats] into the recipient stroke-prone spontaneously hypertensive rats from Glasgow colonies [SHRSP((Gla))], and vice versa. The number of backcross generations required for each strain to achieve complete homozygosity at 83 background genetic markers in a "best" male varied between 3 and 4. Transfer of the region of rat chromosome 2 containing both QTLs from WKY((Gla)) into an SHRSP((Gla)) genetic background lowered both baseline and salt-loaded systolic blood pressure by approximately 20 and approximately 40 mm Hg in male congenic rats compared with the SHRSP parental strain (F=53.4, P<0.005; F=28.0, P< 0.0005, respectively). In contrast, control animals for stowaway heterozygosity presented no deviation from the blood pressure values recorded for the SHRSP((Gla)), indicating that if such heterozygosity exists, its effect on blood pressure is negligible. A reciprocal strategy in which 1 or both QTLs on rat chromosome 2 were transferred from SHRSP((Gla)) into a WKY((Gla)) genetic background resulted in statistically significant but smaller blood pressure increases for 1 of these QTLs. These results confirm the existence of blood pressure QTLs on rat chromosome 2 and demonstrate the applicability of a speed congenic strategy in the rat and emphasize the important role of the genetic background.

  13. Identification of targets of c-Src tyrosine kinase by chemical complementation and phosphoproteomics.

    PubMed

    Ferrando, Isabel Martinez; Chaerkady, Raghothama; Zhong, Jun; Molina, Henrik; Jacob, Harrys K C; Herbst-Robinson, Katie; Dancy, Beverley M; Katju, Vikram; Bose, Ron; Zhang, Jin; Pandey, Akhilesh; Cole, Philip A

    2012-08-01

    The cellular proto-oncogene c-Src is a nonreceptor tyrosine kinase involved in cell growth and cytoskeletal regulation. Despite being dysregulated in a variety of human cancers, its precise functions are not fully understood. Identification of the substrates of c-Src remains a major challenge, because there is no simple way to directly stimulate its activity. Here we combine the chemical rescue of mutant c-Src and global quantitative phosphoproteomics to obtain the first high resolution snapshot of the range of tyrosine phosphorylation events that occur in the cell immediately after specific c-Src stimulation. After enrichment by anti-phosphotyrosine antibodies, we identified 29 potential novel c-Src substrate proteins. Tyrosine phosphopeptide mapping allowed the identification of 382 nonredundant tyrosine phosphopeptides on 213 phosphoproteins. Stable isotope labeling of amino acids in cell culture-based quantitation allowed the detection of 97 nonredundant tyrosine phosphopeptides whose level of phosphorylation is increased by c-Src. A large number of previously uncharacterized c-Src putative protein targets and phosphorylation sites are presented here, a majority of which play key roles in signaling and cytoskeletal networks, particularly in cell adhesion. Integrin signaling and focal adhesion kinase signaling pathway are two of the most altered pathways upon c-Src activation through chemical rescue. In this context, our study revealed the temporal connection between c-Src activation and the GTPase Rap1, known to stimulate integrin-dependent adhesion. Chemical rescue of c-Src provided a tool to dissect the spatiotemporal mechanism of activation of the Rap1 guanine exchange factor, C3G, one of the identified potential c-Src substrates that plays a role in focal adhesion signaling. In addition to unveiling the role of c-Src in the cell and, specifically, in the Crk-C3G-Rap1 pathway, these results exemplify a strategy for obtaining a comprehensive understanding of

  14. Genetic dissection of milk yield traits and mastitis resistance quantitative trait loci on chromosome 20 in dairy cattle.

    PubMed

    Kadri, Naveen K; Guldbrandtsen, Bernt; Lund, Mogens S; Sahana, Goutam

    2015-12-01

    Intense selection to increase milk yield has had negative consequences for mastitis incidence in dairy cattle. Due to low heritability of mastitis resistance and an unfavorable genetic correlation with milk yield, a reduction in mastitis through traditional breeding has been difficult to achieve. Here, we examined quantitative trait loci (QTL) that segregate for clinical mastitis and milk yield on Bos taurus autosome 20 (BTA20) to determine whether both traits are affected by a single polymorphism (pleiotropy) or by multiple closely linked polymorphisms. In the latter but not the former situation, undesirable genetic correlation could potentially be broken by selecting animals that have favorable variants for both traits. First, we performed a within-breed association study using a haplotype-based method in Danish Holstein cattle (HOL). Next, we analyzed Nordic Red dairy cattle (RDC) and Danish Jersey cattle (JER) with the goal of determining whether these QTL identified in Holsteins were segregating across breeds. Genotypes for 12,566 animals (5,966 HOL, 5,458 RDC, and 1,142 JER) were determined by using the Illumina Bovine SNP50 BeadChip (50K; Illumina, San Diego, CA), which identifies 1,568 single nucleotide polymorphisms on BTA20. Data were combined, phased, and clustered into haplotype states, followed by within- and across-breed haplotype-based association analyses using a linear mixed model. Association signals for both clinical mastitis and milk yield peaked in the 26- to 40-Mb region on BTA20 in HOL. Single-variant association analyses were carried out in the QTL region using whole sequence level variants imputed from references of 2,036 HD genotypes (BovineHD BeadChip; Illumina) and 242 whole-genome sequences. The milk QTL were also segregating in RDC and JER on the BTA20-targeted region; however, an indication of differences in the causal factor(s) was observed across breeds. A previously reported F279Y mutation (rs385640152) within the growth hormone

  15. Genetic dissection of milk yield traits and mastitis resistance quantitative trait loci on chromosome 20 in dairy cattle.

    PubMed

    Kadri, Naveen K; Guldbrandtsen, Bernt; Lund, Mogens S; Sahana, Goutam

    2015-12-01

    Intense selection to increase milk yield has had negative consequences for mastitis incidence in dairy cattle. Due to low heritability of mastitis resistance and an unfavorable genetic correlation with milk yield, a reduction in mastitis through traditional breeding has been difficult to achieve. Here, we examined quantitative trait loci (QTL) that segregate for clinical mastitis and milk yield on Bos taurus autosome 20 (BTA20) to determine whether both traits are affected by a single polymorphism (pleiotropy) or by multiple closely linked polymorphisms. In the latter but not the former situation, undesirable genetic correlation could potentially be broken by selecting animals that have favorable variants for both traits. First, we performed a within-breed association study using a haplotype-based method in Danish Holstein cattle (HOL). Next, we analyzed Nordic Red dairy cattle (RDC) and Danish Jersey cattle (JER) with the goal of determining whether these QTL identified in Holsteins were segregating across breeds. Genotypes for 12,566 animals (5,966 HOL, 5,458 RDC, and 1,142 JER) were determined by using the Illumina Bovine SNP50 BeadChip (50K; Illumina, San Diego, CA), which identifies 1,568 single nucleotide polymorphisms on BTA20. Data were combined, phased, and clustered into haplotype states, followed by within- and across-breed haplotype-based association analyses using a linear mixed model. Association signals for both clinical mastitis and milk yield peaked in the 26- to 40-Mb region on BTA20 in HOL. Single-variant association analyses were carried out in the QTL region using whole sequence level variants imputed from references of 2,036 HD genotypes (BovineHD BeadChip; Illumina) and 242 whole-genome sequences. The milk QTL were also segregating in RDC and JER on the BTA20-targeted region; however, an indication of differences in the causal factor(s) was observed across breeds. A previously reported F279Y mutation (rs385640152) within the growth hormone

  16. Computational phosphoproteomics: From identification to localization

    PubMed Central

    Lee, Dave C H; Jones, Andrew R; Hubbard, Simon J

    2015-01-01

    Analysis of the phosphoproteome by MS has become a key technology for the characterization of dynamic regulatory processes in the cell, since kinase and phosphatase action underlie many major biological functions. However, the addition of a phosphate group to a suitable side chain often confounds informatic analysis by generating product ion spectra that are more difficult to interpret (and consequently identify) relative to unmodified peptides. Collectively, these challenges have motivated bioinformaticians to create novel software tools and pipelines to assist in the identification of phosphopeptides in proteomic mixtures, and help pinpoint or “localize” the most likely site of modification in cases where there is ambiguity. Here we review the challenges to be met and the informatics solutions available to address them for phosphoproteomic analysis, as well as highlighting the difficulties associated with using them and the implications for data standards. PMID:25475148

  17. Polyomino Dissections

    ERIC Educational Resources Information Center

    Hohn, Tiina; Liu, Andy

    2012-01-01

    One of Gardner's passions was to introduce puzzles into the classroom. From this point of view, polyomino dissections are an excellent topic. They require little background, provide training in geometric visualization, and mostly they are fun. In this article, we put together a large collection of such puzzles, introduce a new approach in solving…

  18. Sample Preparation for Phosphoproteomic Analysis of Circadian Time Series in Arabidopsis thaliana

    PubMed Central

    Krahmer, Johanna; Hindle, Matthew M.; Martin, Sarah F.; Le Bihan, Thierry; Millar, Andrew J.

    2015-01-01

    Systems biological approaches to study the Arabidopsis thaliana circadian clock have mainly focused on transcriptomics while little is known about the proteome, and even less about posttranslational modifications. Evidence has emerged that posttranslational protein modifications, in particular phosphorylation, play an important role for the clock and its output. Phosphoproteomics is the method of choice for a large-scale approach to gain more knowledge about rhythmic protein phosphorylation. Recent plant phosphoproteomics publications have identified several thousand phosphopeptides. However, the methods used in these studies are very labor-intensive and therefore not suitable to apply to a well-replicated circadian time series. To address this issue, we present and compare different strategies for sample preparation for phosphoproteomics that are compatible with large numbers of samples. Methods are compared regarding number of identifications, variability of quantitation, and functional categorization. We focus on the type of detergent used for protein extraction as well as methods for its removal. We also test a simple two-fraction separation of the protein extract. PMID:25662467

  19. Analysis of the Candida albicans Phosphoproteome

    PubMed Central

    Willger, S. D.; Liu, Z.; Olarte, R. A.; Adamo, M. E.; Myers, L. C.; Kettenbach, A. N.

    2015-01-01

    Candida albicans is an important human fungal pathogen in both immunocompetent and immunocompromised individuals. C. albicans regulation has been studied in many contexts, including morphological transitions, mating competence, biofilm formation, stress resistance, and cell wall synthesis. Analysis of kinase- and phosphatase-deficient mutants has made it clear that protein phosphorylation plays an important role in the regulation of these pathways. In this study, to further our understanding of phosphorylation in C. albicans regulation, we performed a deep analysis of the phosphoproteome in C. albicans. We identified 19,590 unique peptides that corresponded to 15,906 unique phosphosites on 2,896 proteins. The ratios of serine, threonine, and tyrosine phosphosites were 80.01%, 18.11%, and 1.81%, respectively. The majority of proteins (2,111) contained at least two detected phosphorylation sites. Consistent with findings in other fungi, cytoskeletal proteins were among the most highly phosphorylated proteins, and there were differences in Gene Ontology (GO) terms for proteins with serine and threonine versus tyrosine phosphorylation sites. This large-scale analysis identified phosphosites in protein components of Mediator, an important transcriptional coregulatory protein complex. A targeted analysis of the phosphosites in Mediator complex proteins confirmed the large-scale studies, and further in vitro assays identified a subset of these phosphorylations that were catalyzed by Cdk8 (Ssn3), a kinase within the Mediator complex. These data represent the deepest single analysis of a fungal phosphoproteome and lay the groundwork for future analyses of the C. albicans phosphoproteome and specific phosphoproteins. PMID:25750214

  20. Biology teachers' dissection practices and the influences that lead to their adoption: An exploratory research

    NASA Astrophysics Data System (ADS)

    Milano, Regina Nicole

    The lack of resolution in the on-going animal dissection debate inspired this mixed methods study to identify Connecticut secondary biology teachers' dissection practices and the influences that lead to their adoption. Qualitative findings indicate past experiences, managing objections to dissection, school culture, goals of biology teaching and ethics as major influences on dissection practices with 58.4% (n=7) of the sample dissecting and 41.6% not dissecting (n=5). Quantitative findings reveal gender, standards and curriculum, advantages of dissection and experiences as a student as major influences on dissection practices with 71.9% (n=92) of the sample dissecting and 28.1% (n=36) not dissecting. The study concludes that dissection policies are necessary and imminent in Connecticut school districts. Furthermore, it advises teacher-initiated, qualitative and quantitative assessments to expose disparities between student dissection perspectives and their own, prior to conducting dissection. Finally, it provides suggestions for addressing potential differences including administrative involvement.

  1. Toward defining the phosphoproteome of Xenopus laevis embryos

    PubMed Central

    McGivern, Jered V.; Swaney, Danielle L.; Coon, Joshua J.; Sheets, Michael D.

    2010-01-01

    Phosphorylation is universally used for controlling protein function, but knowledge of the phosphoproteome in vertebrate embryos has been limited. However, recent technical advances make it possible to define an organism's phosphoproteome at a more comprehensive level. Xenopus laevis offers established advantages for analyzing the regulation of protein function by phosphorylation. Functionally unbiased, comprehensive information about the Xenopus phosphoproteome would provide a powerful guide for future studies of phosphorylation in a developmental context. To this end, we performed a phosphoproteomic analysis of Xenopus oocytes, eggs, and embryos using recently developed mass spectrometry methods. We identified 1,441 phosphorylation sites present on 654 different Xenopus proteins, including hundreds of previously unknown phosphorylation sites. This approach identified several phosphorylation sites described in the literature and/or evolutionarily conserved in other organisms, validating the data's quality. These data will serve as a powerful resource for the exploration of phosphorylation and protein function within a developmental context. PMID:19384857

  2. Evaluating experimental bias and completeness in comparative phosphoproteomics analysis.

    PubMed

    Boekhorst, Jos; Boersema, Paul J; Tops, Bastiaan B J; van Breukelen, Bas; Heck, Albert J R; Snel, Berend

    2011-01-01

    Unraveling the functional dynamics of phosphorylation networks is a crucial step in understanding the way in which biological networks form a living cell. Recently there has been an enormous increase in the number of measured phosphorylation events. Nevertheless, comparative and integrative analysis of phosphoproteomes is confounded by incomplete coverage and biases introduced by different experimental workflows. As a result, we cannot differentiate whether phosphosites indentified in only one or two samples are the result of condition or species specific phosphorylation, or reflect missing data. Here, we evaluate the impact of incomplete phosphoproteomics datasets on comparative analysis, and we present bioinformatics strategies to quantify the impact of different experimental workflows on measured phosphoproteomes. We show that plotting the saturation in observed phosphosites in replicates provides a reproducible picture of the extent of a particular phosphoproteome. Still, we are still far away from a complete picture of the total human phosphoproteome. The impact of different experimental techniques on the similarity between phosphoproteomes can be estimated by comparing datasets from different experimental pipelines to a common reference. Our results show that comparative analysis is most powerful when datasets have been generated using the same experimental workflow. We show this experimentally by measuring the tyrosine phosphoproteome from Caenorhabditis elegans and comparing it to the tyrosine phosphoproteome of HeLa cells, resulting in an overlap of about 4%. This overlap between very different organisms represents a three-fold increase when compared to dataset of older studies, wherein different workflows were used. The strategies we suggest enable an estimation of the impact of differences in experimental workflows on the overlap between datasets. This will allow us to perform comparative analyses not only on datasets specifically generated for this

  3. Phosphoproteomic analysis of apoptotic hematopoietic stem cells from hemoglobin E/β-thalassemia

    PubMed Central

    2011-01-01

    Background Hemoglobin E/β-thalassemia is particularly common in Southeast Asia and has variable symptoms ranging from mild to severe anemia. Previous investigations demonstrated the remarkable symptoms of β-thalassemia in terms of the acceleration of apoptotic cell death. Ineffective erythropoiesis has been studied in human hematopoietic stem cells, however the distinct apoptotic mechanism was unclear. Methods The phosphoproteome of bone marrow HSCs/CD34+ cells from HbE/β-thalassemic patients was analyzed using IMAC phosphoprotein isolation followed by LC-MS/MS detection. Decyder MS software was used to quantitate differentially expressed proteins in 3 patients and 2 normal donors. The differentially expressed proteins from HSCs/CD34+ cells were compared with HbE/β-thalassemia and normal HSCs. Results A significant change in abundance of 229 phosphoproteins was demonstrated. Importantly, the analysis of the candidate proteins revealed a high abundance of proteins that are commonly found in apoptotic cells including cytochrome C, caspase 6 and apoptosis inducing factors. Moreover, in the HSCs patients a significant increase was observed in a specific type of phosphoserine/threonine binding protein, which is known to act as an important signal mediator for the regulation of cell survival and apoptosis in HbE/β-thalassemia. Conclusions Our study used a novel method to investigate proteins that influence a particular pathway in a given disease or physiological condition. Ultimately, phosphoproteome profiling in HbE/β-thalassemic stem cells is an effective method to further investigate the cell death mechanism of ineffective erythropoiesis in β-thalassemia. Our report provides a comprehensive phosphoproteome, an important resource for the study of ineffective erythropoiesis and developing therapies for HbE/β-thalassemia. PMID:21702968

  4. Phosphoproteomic analysis of the response of maize leaves to drought, heat and their combination stress

    PubMed Central

    Hu, Xiuli; Wu, Liuji; Zhao, Feiyun; Zhang, Dayong; Li, Nana; Zhu, Guohui; Li, Chaohao; Wang, Wei

    2015-01-01

    Drought and heat stress, especially their combination, greatly affect crop production. Many studies have described transcriptome, proteome and phosphoproteome changes in response of plants to drought or heat stress. However, the study about the phosphoproteomic changes in response of crops to the combination stress is scare. To understand the mechanism of maize responses to the drought and heat combination stress, phosphoproteomic analysis was performed on maize leaves by using multiplex iTRAQ-based quantitative proteomic and LC-MS/MS methods. Five-leaf-stage maize was subjected to drought, heat or their combination, and the leaves were collected. Globally, heat, drought and the combined stress significantly changed the phosphorylation levels of 172, 149, and 144 phosphopeptides, respectively. These phosphopeptides corresponded to 282 proteins. Among them, 23 only responded to the combined stress and could not be predicted from their responses to single stressors; 30 and 75 only responded to drought and heat, respectively. Notably, 19 proteins were phosphorylated on different sites in response to the single and combination stresses. Of the seven significantly enriched phosphorylation motifs identified, two were common for all stresses, two were common for heat and the combined stress, and one was specific to the combined stress. The signaling pathways in which the phosphoproteins were involved clearly differed among the three stresses. Functional characterization of the phosphoproteins and the pathways identified here could lead to new targets for the enhancement of crop stress tolerance, which will be particularly important in the face of climate change and the increasing prevalence of abiotic stressors. PMID:25999967

  5. Novel Host Proteins and Signaling Pathways in Enteropathogenic E. coli Pathogenesis Identified by Global Phosphoproteome Analysis*

    PubMed Central

    Scholz, Roland; Imami, Koshi; Scott, Nichollas E.; Trimble, William S.; Foster, Leonard J.; Finlay, B. Brett

    2015-01-01

    Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (T3SS) to directly translocate effector proteins into host cells where they play a pivotal role in subverting host cell signaling needed for disease. However, our knowledge of how EPEC affects host protein phosphorylation is limited to a few individual protein studies. We employed a quantitative proteomics approach to globally map alterations in the host phosphoproteome during EPEC infection. By characterizing host phosphorylation events at various time points throughout infection, we examined how EPEC dynamically impacts the host phosphoproteome over time. This experimental setup also enabled identification of T3SS-dependent and -independent changes in host phosphorylation. Specifically, T3SS-regulated events affected various cellular processes that are known EPEC targets, including cytoskeletal organization, immune signaling, and intracellular trafficking. However, the involvement of phosphorylation in these events has thus far been poorly studied. We confirmed the MAPK family as an established key host player, showed its central role in signal transduction during EPEC infection, and extended the repertoire of known signaling hubs with previously unrecognized proteins, including TPD52, CIN85, EPHA2, and HSP27. We identified altered phosphorylation of known EPEC targets, such as cofilin, where the involvement of phosphorylation has so far been undefined, thus providing novel mechanistic insights into the roles of these proteins in EPEC infection. An overlap of regulated proteins, especially those that are cytoskeleton-associated, was observed when compared with the phosphoproteome of Shigella-infected cells. We determined the biological relevance of the phosphorylation of a novel protein in EPEC pathogenesis, septin-9 (SEPT9). Both siRNA knockdown and a phosphorylation-impaired SEPT9 mutant decreased bacterial adherence and EPEC-mediated cell death. In contrast, a phosphorylation

  6. Phosphoproteomic analysis of wild-type and antimony-resistant Leishmania braziliensis lines by 2D-DIGE technology.

    PubMed

    Moreira, Douglas de Souza; Pescher, Pascale; Laurent, Christine; Lenormand, Pascal; Späth, Gerald F; Murta, Silvane M F

    2015-09-01

    Protein phosphorylation is one of the most studied post-translational modifications that is involved in different cellular events in Leishmania. In this study, we performed a comparative phosphoproteomics analysis of potassium antimonyl tartrate (SbIII)-resistant and -susceptible lines of Leishmania braziliensis using a 2D-DIGE approach followed by MS. In order to investigate the differential phosphoprotein abundance associated with the drug-induced stress response and SbIII-resistance mechanisms, we compared nontreated and SbIII-treated samples of each line. Pair wise comparisons revealed a total of 116 spots that showed a statistically significant difference in phosphoprotein abundance, including 11 and 34 spots specifically correlated with drug treatment and resistance, respectively. We identified 48 different proteins distributed into seven biological process categories. The category "protein folding/chaperones and stress response" is mainly implicated in response to SbIII treatment, while the categories "antioxidant/detoxification," "metabolic process," "RNA/DNA processing," and "protein biosynthesis" are modulated in the case of antimony resistance. Multiple sequence alignments were performed to validate the conservation of phosphorylated residues in nine proteins identified here. Western blot assays were carried out to validate the quantitative phosphoproteome analysis. The results revealed differential expression level of three phosphoproteins in the lines analyzed. This novel study allowed us to profile the L. braziliensis phosphoproteome, identifying several potential candidates for biochemical or signaling networks associated with antimony resistance phenotype in this parasite.

  7. Quantitative phosphoproteome analysis of embryonic stem cell differentiation toward blood

    PubMed Central

    Piazzi, Manuela; Williamson, Andrew; Lee, Chia-Fang; Pearson, Stella; Lacaud, Georges; Kouskoff, Valerie; McCubrey, James A.; Cocco, Lucio; Whetton, Anthony D.

    2015-01-01

    Murine embryonic stem (ES) cells can differentiate in vitro into three germ layers (endodermic, mesodermic, ectodermic). Studies on the differentiation of these cells to specific early differentiation stages has been aided by an ES cell line carrying the Green Fluorescent Protein (GFP) targeted to the Brachyury (Bry) locus which marks mesoderm commitment. Furthermore, expression of the Vascular Endothelial Growth Factor receptor 2 (Flk1) along with Bry defines hemangioblast commitment. Isobaric-tag for relative and absolute quantification (iTRAQTM) and phosphopeptide enrichment coupled to liquid chromatography separation and mass spectrometry allow the study of phosphorylation changes occurring at different stages of ES cell development using Bry and Flk1 expression respectively. We identified and relatively quantified 37 phosphoentities which are modulated during mesoderm-induced ES cells differentiation, comparing epiblast-like, early mesoderm and hemangioblast-enriched cells. Among the proteins differentially phosphorylated toward mesoderm differentiation were: the epigenetic regulator Dnmt3b, the protein kinase GSK3b, the chromatin remodeling factor Smarcc1, the transcription factor Utf1; as well as protein specifically related to stem cell differentiation, as Eomes, Hmga2, Ints1 and Rif1. As most key factors regulating early hematopoietic development have also been implicated in various types of leukemia, understanding the post-translational modifications driving their regulation during normal development could result in a better comprehension of their roles during abnormal hematopoiesis in leukemia. PMID:25890499

  8. Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants

    PubMed Central

    Greenwood, Edward JD; Matheson, Nicholas J; Wals, Kim; van den Boomen, Dick JH; Antrobus, Robin; Williamson, James C; Lehner, Paul J

    2016-01-01

    Viruses manipulate host factors to enhance their replication and evade cellular restriction. We used multiplex tandem mass tag (TMT)-based whole cell proteomics to perform a comprehensive time course analysis of >6500 viral and cellular proteins during HIV infection. To enable specific functional predictions, we categorized cellular proteins regulated by HIV according to their patterns of temporal expression. We focussed on proteins depleted with similar kinetics to APOBEC3C, and found the viral accessory protein Vif to be necessary and sufficient for CUL5-dependent proteasomal degradation of all members of the B56 family of regulatory subunits of the key cellular phosphatase PP2A (PPP2R5A-E). Quantitative phosphoproteomic analysis of HIV-infected cells confirmed Vif-dependent hyperphosphorylation of >200 cellular proteins, particularly substrates of the aurora kinases. The ability of Vif to target PPP2R5 subunits is found in primate and non-primate lentiviral lineages, and remodeling of the cellular phosphoproteome is therefore a second ancient and conserved Vif function. DOI: http://dx.doi.org/10.7554/eLife.18296.001 PMID:27690223

  9. Phosphoproteomic analysis reveals compensatory effects in the piriform cortex of VX nerve agent exposed rats.

    PubMed

    Nirujogi, Raja Sekhar; Wright, James D; Manda, Srikanth S; Zhong, Jun; Na, Chan Hyun; Meyerhoff, James; Benton, Bernard; Jabbour, Rabih; Willis, Kristen; Kim, Min-Sik; Pandey, Akhilesh; Sekowski, Jennifer W

    2015-01-01

    To gain insights into the toxicity induced by the nerve agent VX, an MS-based phosphoproteomic analysis was carried out on the piriform cortex region of brains from VX-treated rats. Using isobaric tag based TMT labeling followed by titanium dioxide enrichment strategy, we identified 9975 unique phosphosites derived from 3287 phosphoproteins. Temporal changes in the phosphorylation status of peptides were observed over a time period of 24 h in rats exposed to a 1× LD50, intravenous (i.v.) dose with the most notable changes occurring at the 1 h postexposure time point. Five major functional classes of proteins exhibited changes in their phosphorylation status: (i) ion channels/transporters, including ATPases, (ii) kinases/phosphatases, (iii) GTPases, (iv) structural proteins, and (v) transcriptional regulatory proteins. This study is the first quantitative phosphoproteomic analysis of VX toxicity in the brain. Understanding the toxicity and compensatory signaling mechanisms will improve the understanding of the complex toxicity of VX in the brain and aid in the elucidation of novel molecular targets that would be important for development of improved countermeasures. All MS data have been deposited in the ProteomeXchange with identifier PXD001184 (http://proteomecentral.proteomexchange.org/dataset/PXD001184).

  10. Phosphoproteomic profiling of mouse primary HSPCs reveals new regulators of HSPC mobilization

    PubMed Central

    Ficarro, Scott B.; Hutchinson, John N.; Csepanyi-Komi, Roland; Nguyen, Phi T.; Wisniewski, Eva; Sullivan, Jessica; Hofmann, Oliver; Ligeti, Erzsebet; Marto, Jarrod A.; Wagers, Amy J.

    2016-01-01

    Protein phosphorylation is a central mechanism of signal transduction that both positively and negatively regulates protein function. Large-scale studies of the dynamic phosphorylation states of cell signaling systems have been applied extensively in cell lines and whole tissues to reveal critical regulatory networks, and candidate-based evaluations of phosphorylation in rare cell populations have also been informative. However, application of comprehensive profiling technologies to adult stem cell and progenitor populations has been challenging, due in large part to the scarcity of such cells in adult tissues. Here, we combine multicolor flow cytometry with highly efficient 3-dimensional high performance liquid chromatography/mass spectrometry to enable quantitative phosphoproteomic analysis from 200 000 highly purified primary mouse hematopoietic stem and progenitor cells (HSPCs). Using this platform, we identify ARHGAP25 as a novel regulator of HSPC mobilization and demonstrate that ARHGAP25 phosphorylation at serine 363 is an important modulator of its function. Our approach provides a robust platform for large-scale phosphoproteomic analyses performed with limited numbers of rare progenitor cells. Data from our study comprises a new resource for understanding the molecular signaling networks that underlie hematopoietic stem cell mobilization. PMID:27365422

  11. Versatile nanocomposites in phosphoproteomics: a review.

    PubMed

    Najam-ul-Haq, Muhammad; Jabeen, Fahmida; Hussain, Dilshad; Saeed, Adeela; Musharraf, Syed Ghulam; Huck, Christian W; Bonn, Günther K

    2012-10-17

    Protein phosphorylation is one of the most important post-translational modifications. Phosphorylated peptides are present in low abundance in blood serum but play a vital role in regulatory mechanisms and may serve as casual factors in diseases. The enrichment and analysis of phosphorylated peptides directly from human serum and mapping the phosphorylation sites is a challenging task. Versatile nanocomposites of different materials have been synthesized using simple but efficient methodologies for their enrichment. The nanocomposites include magnetic, coated, embedded as well as chemically derivatized materials. Different base materials such as polymers, carbon based and metal oxides are used. The comparison of nanocomposites with respective nanoparticles provides sufficient facts about their efficiency in terms of loading capacity and capture efficiency. The cost for preparing them is low and they hold great promise to be used as chromatographic materials for phosphopeptide enrichment. This review gives an overview of different nanocomposites in phosphoproteomics, discussing the improved efficiency than the individual counterparts and highlighting their significance in phosphopeptide enrichment. PMID:22986130

  12. Phosphoproteomic analysis of aged skeletal muscle.

    PubMed

    Gannon, Joan; Staunton, Lisa; O'Connell, Kathleen; Doran, Philip; Ohlendieck, Kay

    2008-07-01

    One of the most important post-translational modifications is represented by phosphorylation on tyrosine, threonine and serine residues. Since abnormal phosphorylation is associated with various pathologies, it was of interest to perform a phosphoproteomic profiling of age-related skeletal muscle degeneration. We used the fluorescent phospho-specific Pro-Q Diamond dye to determine whether changes in the overall phosphorylation of the soluble skeletal muscle proteome differs significantly between young adult and senescent fibres. As an established model system of sarcopenia, we employed 30-month-old rat gastrocnemius fibres. Following the mass spectrometric identification of 59 major 2-D phosphoprotein landmark spots, the fluorescent dye staining survey revealed that 22 muscle proteins showed a differential expression pattern between 3-month- and 30-month-old muscle. Increased phosphorylation levels were shown for myosin light chain 2, tropomyosin alpha, lactate dehydrogenase, desmin, actin, albumin and aconitase. In contrast, decreased phospho-specific dye binding was observed for cytochrome c oxidase, creatine kinase and enolase. Thus, aging-induced alterations in phosphoproteins appear to involve the contractile machinery and the cytoskeleton, as well as the cytosolic and mitochondrial metabolism. This confirms that sarcopenia of old age is a complex neuromuscular pathology that is associated with drastic changes in the abundance and structure of key muscle proteins. PMID:18575773

  13. Dissecting Classroom Ethics.

    ERIC Educational Resources Information Center

    Allchin, Douglas

    1991-01-01

    Described are activities that lead to values clarification. Issues such as dissection, bioengineering, birth control, medical resources, and death are discussed. Included is a student questionnaire on the subject of dissection and the use of animals in laboratories. (KR)

  14. The Problems of Dissection.

    ERIC Educational Resources Information Center

    Davis, Pat

    1997-01-01

    Describes some problems of classroom dissection including the cruelty that animals destined for the laboratory suffer. Discusses the multilevel approach that the National Anti-Vivisection Society (NAVS) has developed to address the problems of animal dissection such as offering a dissection hotline, exhibiting at science teacher conferences, and…

  15. Perspectives of comprehensive phosphoproteome analysis using shotgun strategy.

    PubMed

    Wang, Fangjun; Song, Chunxia; Cheng, Kai; Jiang, Xinning; Ye, Mingliang; Zou, Hanfa

    2011-11-01

    Protein phosphorylation is a ubiquitous post-translational modification that regulates almost all cellular processes. The analysis of protein phosphorylation is challenging due to the high dynamic range and low abundance natures of the analyte. Shotgun based proteomics has emerged as a very useful platform to achieve a comprehensive phosphoproteome analysis in considerable depth. In the past few years, significant breakthroughs on the large scale phosphorylation analysis have been witnessed along with the great development of related technologies. The combination of effective enrichment materials, refined analysis workflows, new type of powerful mass spectrometers, and sophisticated bioinformatic tools greatly boost the performance of comprehensive phosphoproteome analysis. In this Perspective, we briefly reviewed recent technological developments on the enrichment materials, prefractionation workflows, and different mass spectrometry fragmentation modes as well as software tools for phosphoproteome identification and quantification. Then, we described the current challenges and potential directions for the future of comprehensive phosphoproteome analysis. We also provide perspectives on how to further improve the performance of related analysis methods and technologies.

  16. Phosphoproteomics Analysis of Endometrium in Women with or without Endometriosis

    PubMed Central

    Xu, Hong-Mei; Deng, Hai-Teng; Liu, Chong-Dong; Chen, Yu-Ling; Zhang, Zhen-Yu

    2015-01-01

    Background: The molecular mechanisms underlying the endometriosis are still not completely understood. In order to test the hypothesis that the approaches in phosphoproteomics might contribute to the identification of key biomarkers to assess disease pathogenesis and drug targets, we carried out a phosphoproteomics analysis of human endometrium. Methods: A large-scale differential phosphoproteome analysis, using peptide enrichment of titanium dioxide purify and sequential elution from immobilized metal affinity chromatography with linear trap quadrupole-tandem mass spectrometry, was performed in endometrium tissues from 8 women with or without endometriosis. Results: The phosphorylation profiling of endometrium from endometriosis patients had been obtained, and found that identified 516 proteins were modified at phosphorylation level during endometriosis. Gene ontology annotation analysis showed that these proteins were enriched in cellular processes of binding and catalytic activity. Further pathway analysis showed that ribosome pathway and focal adhesion pathway were the top two pathways, which might be deregulated during the development of endometriosis. Conclusions: That large-scale phosphoproteome quantification has been successfully identified in endometrium tissues of women with or without endometriosis will provide new insights to understand the molecular mechanisms of the development of endometriosis. PMID:26415800

  17. Hippocampal phosphoproteomics of F344 rats exposed to 1-bromopropane

    SciTech Connect

    Huang, Zhenlie; Ichihara, Sahoko; Oikawa, Shinji; Chang, Jie; Zhang, Lingyi; Hu, Shijie; Huang, Hanlin; Ichihara, Gaku

    2015-01-15

    1-Bromopropane (1-BP) is neurotoxic in both experimental animals and human. To identify phosphorylated modification on the unrecognized post-translational modifications of proteins and investigate their role in 1-BP-induced neurotoxicity, changes in hippocampal phosphoprotein expression levels were analyzed quantitatively in male F344 rats exposed to 1-BP inhalation at 0, 400, or 1000 ppm for 8 h/day for 1 or 4 weeks. Hippocampal protein extracts were analyzed qualitatively and quantitatively by Pro-Q Diamond gel staining and SYPRO Ruby staining coupled with two-dimensional difference in gel electrophoresis (2D-DIGE), respectively, as well as by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) to identify phosphoproteins. Changes in selected proteins were further confirmed by Manganese II (Mn{sup 2+})-Phos-tag SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Bax and cytochrome c protein levels were determined by western blotting. Pro-Q Diamond gel staining combined with 2D-DIGE identified 26 phosphoprotein spots (p < 0.05), and MALDI-TOF/MS identified 18 up-regulated proteins and 8 down-regulated proteins. These proteins are involved in the biological process of response to stimuli, metabolic processes, and apoptosis signaling. Changes in the expression of phosphorylated 14-3-3 θ were further confirmed by Mn{sup 2+}-Phos-tag SDS-PAGE. Western blotting showed overexpression of Bax protein in the mitochondria with down-regulation in the cytoplasm, whereas cytochrome c expression was high in the cytoplasm but low in the mitochondria after 1-BP exposure. Our results suggest that the pathogenesis of 1-BP-induced hippocampal damage involves inhibition of antiapoptosis process. Phosphoproteins identified in this study can potentially serve as biomarkers for 1-BP-induced neurotoxicity. - Highlights: • 1-BP modified hippocampal phosphoproteome in rat and 23 altered proteins were identified. • 1-BP changed phosphorylation

  18. Spatial proteomic and phospho-proteomic organization in three prototypical cell migration modes

    PubMed Central

    2014-01-01

    Background Tight spatio-temporal signaling of cytoskeletal and adhesion dynamics is required for localized membrane protrusion that drives directed cell migration. Different ensembles of proteins are therefore likely to get recruited and phosphorylated in membrane protrusions in response to specific cues. Results Here, we use an assay that allows to biochemically purify extending protrusions of cells migrating in response to three prototypical receptors: integrins, recepor tyrosine kinases and G-coupled protein receptors. Using quantitative proteomics and phospho-proteomics approaches, we provide evidence for the existence of cue-specific, spatially distinct protein networks in the different cell migration modes. Conclusions The integrated analysis of the large-scale experimental data with protein information from databases allows us to understand some emergent properties of spatial regulation of signaling during cell migration. This provides the cell migration community with a large-scale view of the distribution of proteins and phospho-proteins regulating directed cell migration. PMID:24987309

  19. Battle through Signaling between Wheat and the Fungal Pathogen Septoria tritici Revealed by Proteomics and Phosphoproteomics*

    PubMed Central

    Yang, Fen; Melo-Braga, Marcella N.; Larsen, Martin R.; Jørgensen, Hans J. L.; Palmisano, Giuseppe

    2013-01-01

    The fungus Septoria tritici causes the disease septoria tritici blotch in wheat, one of the most economically devastating foliar diseases in this crop. To investigate signaling events and defense responses in the wheat–S. tritici interaction, we performed a time-course study of S. tritici infection in resistant and susceptible wheat using quantitative proteomics and phosphoproteomics, with special emphasis on the initial biotrophic phase of interactions. Our study revealed an accumulation of defense and stress-related proteins, suppression of photosynthesis, and changes in sugar metabolism during compatible and incompatible interactions. However, differential regulation of the phosphorylation status of signaling proteins, transcription and translation regulators, and membrane-associated proteins was observed between two interactions. The proteomic data were correlated with a more rapid or stronger accumulation of signal molecules, including calcium, H2O2, NO, and sugars, in the resistant than in the susceptible cultivar in response to the infection. Additionally, 31 proteins and 5 phosphoproteins from the pathogen were identified, including metabolic proteins and signaling proteins such as GTP-binding proteins, 14–3-3 proteins, and calcium-binding proteins. Quantitative PCR analysis showed the expression of fungal signaling genes and genes encoding a superoxide dismutase and cell-wall degrading enzymes. These results indicate roles of signaling, antioxidative stress mechanisms, and nutrient acquisition in facilitating the initial symptomless growth. Taken in its entirety, our dataset suggests interplay between the plant and S. tritici through complex signaling networks and downstream molecular events. Resistance is likely related to several rapidly and intensively triggered signal transduction cascades resulting in a multiple-level activation of transcription and translation processes of defense responses. Our sensitive approaches and model provide a

  20. Data set from a comprehensive phosphoproteomic analysis of rice variety IRBB5 in response to bacterial blight

    PubMed Central

    Hou, Yuxuan; Tong, Xiaohong; Wang, Yifeng; Qiu, Jiehua; Li, Zhiyong; Zhang, Wen; Huang, Shiwen; Zhang, Jian

    2015-01-01

    Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) has become one of the most devastating diseases for rice, a major food source for over half of the world populations. To investigate the roles of protein phosphorylation in rice bacterial blight resistance, a quantitative phosphoproteomic study was conducted in rice variety IRBB5 at 0 h and 24 h after Xoo infection. 2367 and 2223 phosphosites on 1334 and 1297 representative proteins were identified in 0 h and 24 h after Xoo infection, respectively, out of which 762 proteins were found to be differentially phosphorylated. In associated with the published article “A comprehensive quantitative phosphoproteome analysis of rice in response to bacterial blight” in BMC Plant Biology (Hou et al., 2015) [1], this dataset article provided the detailed information of experimental designing, methods, features as well as the raw data of mass spectrometry (MS) identification. The MS proteomics data could be fully accessed from the ProteomeXchange Consortium with the dataset identifier PXD002222. PMID:26862573

  1. Phosphoproteome Integration Reveals Patient-Specific Networks in Prostate Cancer.

    PubMed

    Drake, Justin M; Paull, Evan O; Graham, Nicholas A; Lee, John K; Smith, Bryan A; Titz, Bjoern; Stoyanova, Tanya; Faltermeier, Claire M; Uzunangelov, Vladislav; Carlin, Daniel E; Fleming, Daniel Teo; Wong, Christopher K; Newton, Yulia; Sudha, Sud; Vashisht, Ajay A; Huang, Jiaoti; Wohlschlegel, James A; Graeber, Thomas G; Witte, Owen N; Stuart, Joshua M

    2016-08-11

    We used clinical tissue from lethal metastatic castration-resistant prostate cancer (CRPC) patients obtained at rapid autopsy to evaluate diverse genomic, transcriptomic, and phosphoproteomic datasets for pathway analysis. Using Tied Diffusion through Interacting Events (TieDIE), we integrated differentially expressed master transcriptional regulators, functionally mutated genes, and differentially activated kinases in CRPC tissues to synthesize a robust signaling network consisting of druggable kinase pathways. Using MSigDB hallmark gene sets, six major signaling pathways with phosphorylation of several key residues were significantly enriched in CRPC tumors after incorporation of phosphoproteomic data. Individual autopsy profiles developed using these hallmarks revealed clinically relevant pathway information potentially suitable for patient stratification and targeted therapies in late stage prostate cancer. Here, we describe phosphorylation-based cancer hallmarks using integrated personalized signatures (pCHIPS) that shed light on the diversity of activated signaling pathways in metastatic CRPC while providing an integrative, pathway-based reference for drug prioritization in individual patients. PMID:27499020

  2. "Hands-Off" Dissection?

    ERIC Educational Resources Information Center

    Allchin, Douglas

    2005-01-01

    Computer programs and models are used to express respect for life by not sacrificing any animal but these alternatives might be deeply flawed. Alternatives to dissection are perverse alternatives that tend to preserve the features of inappropriate dissections like destructiveness, reductionism and objectification.

  3. Reliable PCR quantitation of estrogen, progesterone and ERBB2 receptor mRNA from formalin-fixed, paraffin-embedded tissue is independent of prior macro-dissection.

    PubMed

    Tramm, Trine; Hennig, Guido; Kyndi, Marianne; Alsner, Jan; Sørensen, Flemming Brandt; Myhre, Simen; Sørlie, Therese; Overgaard, Jens

    2013-12-01

    Gene expression analysis on messenger RNA (mRNA) purified from formalin-fixed, paraffin-embedded tissue is increasingly used for research purposes. Tissue heterogeneity may question specificity and interpretation of results from mRNA isolated from a whole slide section, and thresholds for minimal tumor content in the paraffin block or macrodissection are used to avoid contamination from non-neoplastic tissue. The aim was to test if mRNA from tissue surrounding breast cancer affected quantification of estrogen receptor α (ESR1), progesterone receptor (PGR) and human epidermal growth factor receptor 2 (ERBB2), by comparing gene expression from whole slide and tumor-enriched sections, and correlating gene expression from whole slide sections with corresponding immunohistochemistry. Gene expression, based on mRNA extracted from a training set (36 paraffin blocks) and two validation sets (133 + 1,083 blocks), were determined by quantitative reverse transcription polymerase chain reaction for all samples, as well as by microarray for 133 validation samples. In the training set, agreement between high vs. low mRNA expression from whole slide and tumor-enriched sections was absolute for ESR1 and ERBB2, and 83 % for PGR. Overall agreements, when comparing mRNA expression to immunohistochemistry, were 100 % (ERBB2), 89 % (ESR1) and 83 % (PGR), which was confirmed in the validation sets. Percentage of tumor in the sections did not influence the results. In conclusion, reliable quantification of ESR1, PGR and ERBB2 mRNA expression can be obtained from a whole slide section, and correlates well with immunohistochemistry. Prior removal of surrounding tissue was found to be unnecessary even with minimal tumor content in the section. PMID:24100522

  4. Phosphoproteomic analysis of seed maturation in Arabidopsis, rapeseed, and soybean.

    PubMed

    Meyer, Louis J; Gao, Jianjiong; Xu, Dong; Thelen, Jay J

    2012-05-01

    To characterize protein phosphorylation in developing seed, a large-scale, mass spectrometry-based phosphoproteomic study was performed on whole seeds at five sequential stages of development in soybean (Glycine max), rapeseed (Brassica napus), and Arabidopsis (Arabidopsis thaliana). Phosphopeptides were enriched from 0.5 mg of total peptides using a combined strategy of immobilized metal affinity and metal oxide affinity chromatography. Enriched phosphopeptides were analyzed by Orbitrap tandem mass spectrometry and mass spectra mined against cognate genome or cDNA databases in both forward and randomized orientations, the latter to calculate false discovery rate. We identified a total of 2,001 phosphopeptides containing 1,026 unambiguous phosphorylation sites from 956 proteins, with an average false discovery rate of 0.78% for the entire study. The entire data set was uploaded into the Plant Protein Phosphorylation Database (www.p3db.org), including all meta-data and annotated spectra. The Plant Protein Phosphorylation Database is a portal for all plant phosphorylation data and allows for homology-based querying of experimentally determined phosphosites. Comparisons with other large-scale phosphoproteomic studies determined that 652 of the phosphoproteins are novel to this study. The unique proteins fall into several Gene Ontology categories, some of which are overrepresented in our study as well as other large-scale phosphoproteomic studies, including metabolic process and RNA binding; other categories are only overrepresented in our study, like embryonic development. This investigation shows the importance of analyzing multiple plants and plant organs to comprehensively map the complete plant phosphoproteome.

  5. Proteomics and Phosphoproteomics Analysis of Human Lens Fiber Cell Membranes

    PubMed Central

    Wang, Zhen; Han, Jun; David, Larry L.; Schey, Kevin L.

    2013-01-01

    Purpose. The human lens fiber cell insoluble membrane fraction contains important membrane proteins, cytoskeletal proteins, and cytosolic proteins that are strongly associated with the membrane. The purpose of this study was to characterize the lens fiber cell membrane proteome and phosphoproteome from human lenses. Methods. HPLC-mass spectrometry–based multidimensional protein identification technology (MudPIT), without or with phosphopeptide enrichment, was applied to study the proteome and phosphoproteome of lens fiber cell membranes, respectively. Results. In total, 951 proteins were identified, including 379 integral membrane and membrane-associated proteins. Enriched gene categories and pathways based on the proteomic analysis include carbohydrate metabolism (glycolysis/gluconeogenesis, pentose phosphate pathway, pyruvate metabolism), proteasome, cell-cell signaling and communication (GTP binding, gap junction, focal adhesion), glutathione metabolism, and actin regulation. The combination of TiO2 phosphopeptide enrichment and MudPIT analysis revealed 855 phosphorylation sites on 271 proteins, including 455 phosphorylation sites that have not been previously identified. PKA, PKC, CKII, p38MAPK, and RSK are predicted as the major kinases for phosphorylation on the sites identified in the human lens membrane fraction. Conclusions. The results presented herein significantly expand the characterized proteome and phosphoproteome of the human lens fiber cell and provide a valuable reference for future research in studies of lens development and disease. PMID:23349431

  6. Genetic Dissection of Quantitative Trait Loci for Hemostasis and Thrombosis on Mouse Chromosomes 11 and 5 Using Congenic and Subcongenic Strains

    PubMed Central

    Hoover-Plow, Jane; Sa, Qila; Huang, Menggui; Grondolsky, Jessica

    2013-01-01

    Susceptibility to thrombosis varies in human populations as well as many inbred mouse strains. Only a small portion of this variation has been identified, suggesting that there are unknown modifier genes. The objective of this study was to narrow the quantitative trait locus (QTL) intervals previously identified for hemostasis and thrombosis on mouse distal chromosome 11 (Hmtb6) and on chromosome 5 (Hmtb4 and Hmtb5). In a tail bleeding/rebleeding assay, a reporter assay for hemostasis and thrombosis, subcongenic strain (6A-2) had longer clot stability time than did C57BL/6J (B6) mice but a similar time to the B6-Chr11A/J consomic mice, confirming the Hmtb6 phenotype. Six congenic and subcongenic strains were constructed for chromosome 5, and the congenic strain, 2A-1, containing the shortest A/J interval (16.6 cM, 26.6 Mbp) in the Hmtb4 region, had prolonged clot stability time compared to B6 mice. In the 3A-2 and CSS-5 mice bleeding time was shorter than for B6, mice confirming the Hmtb5 QTL. An increase in bleeding time was identified in another congenic strain (3A-1) with A/J interval (24.8 cM, 32.9 Mbp) in the proximal region of chromosome 5, confirming a QTL for bleeding previously mapped to that region and designated as Hmtb10. The subcongenic strain 4A-2 with the A/J fragment in the proximal region had a long occlusion time of the carotid artery after ferric chloride injury and reduced dilation after injury to the abdominal aorta compared to B6 mice, suggesting an additional locus in the proximal region, which was designated Hmtb11 (5 cM, 21.4 Mbp). CSS-17 mice crossed with congenic strains, 3A-1 and 3A-2, modified tail bleeding. Using congenic and subcongenic analysis, candidate genes previously identified and novel genes were identified as modifiers of hemostasis and thrombosis in each of the loci Hmtb6, Hmtb4, Hmtb10, and Hmtb11. PMID:24147020

  7. Phosphoproteomic analysis of induced resistance reveals activation of signal transduction processes by beneficial and pathogenic interaction in grapevine.

    PubMed

    Perazzolli, Michele; Palmieri, Maria Cristina; Matafora, Vittoria; Bachi, Angela; Pertot, Ilaria

    2016-05-20

    Protein phosphorylation regulates several key processes of the plant immune system. Protein kinases and phosphatases are pivotal regulators of defense mechanisms elicited by resistance inducers. However, the phosphorylation cascades that trigger the induced resistance mechanisms in plants have not yet been deeply investigated. The beneficial fungus Trichoderma harzianum T39 (T39) induces resistance against grapevine downy mildew (Plasmopara viticola), but its efficacy could be further improved by a better understanding of the cellular regulations involved. We investigated quantitative changes in the grapevine phosphoproteome during T39-induced resistance to get an overview of regulatory mechanisms of downy mildew resistance. Immunodetection experiments revealed activation of the 45 and 49kDa kinases by T39 treatment both before and after pathogen inoculation, and the phosphoproteomic analysis identified 103 phosphopeptides that were significantly affected by the phosphorylation cascades during T39-induced resistance. Peptides affected by T39 treatment showed comparable phosphorylation levels after P. viticola inoculation, indicating activation of the microbial recognition machinery before pathogen infection. Phosphorylation profiles of proteins related to photosynthetic processes and protein ubiquitination indicated a partial overlap of cellular responses in T39-treated and control plants. However, phosphorylation changes of proteins involved in response to stimuli, signal transduction, hormone signaling, gene expression regulation, and RNA metabolism were exclusively elicited by P. viticola inoculation in T39-treated plants. These results highlighted the relevance of phosphorylation changes during T39-induced resistance and identified key regulator candidates of the grapevine defense against downy mildew. PMID:27010348

  8. GPM Dissects Typhoon Hagupit

    NASA Video Gallery

    NASA/JAXA's GPM Dissects Typhoon Hagupit Animation revealing a swath of NASA/JAXA's Global Precipitation Measurement (GPM) mission's Core Observatory GMI precipitation rates over Typhoon Hagupit. A...

  9. Parametric binary dissection

    NASA Technical Reports Server (NTRS)

    Bokhari, Shahid H.; Crockett, Thomas W.; Nicol, David M.

    1993-01-01

    Binary dissection is widely used to partition non-uniform domains over parallel computers. This algorithm does not consider the perimeter, surface area, or aspect ratio of the regions being generated and can yield decompositions that have poor communication to computation ratio. Parametric Binary Dissection (PBD) is a new algorithm in which each cut is chosen to minimize load + lambda x(shape). In a 2 (or 3) dimensional problem, load is the amount of computation to be performed in a subregion and shape could refer to the perimeter (respectively surface) of that subregion. Shape is a measure of communication overhead and the parameter permits us to trade off load imbalance against communication overhead. When A is zero, the algorithm reduces to plain binary dissection. This algorithm can be used to partition graphs embedded in 2 or 3-d. Load is the number of nodes in a subregion, shape the number of edges that leave that subregion, and lambda the ratio of time to communicate over an edge to the time to compute at a node. An algorithm is presented that finds the depth d parametric dissection of an embedded graph with n vertices and e edges in O(max(n log n, de)) time, which is an improvement over the O(dn log n) time of plain binary dissection. Parallel versions of this algorithm are also presented; the best of these requires O((n/p) log(sup 3)p) time on a p processor hypercube, assuming graphs of bounded degree. How PBD is applied to 3-d unstructured meshes and yields partitions that are better than those obtained by plain dissection is described. Its application to the color image quantization problem is also discussed, in which samples in a high-resolution color space are mapped onto a lower resolution space in a way that minimizes the color error.

  10. Is dissection humane?

    PubMed Central

    Hasan, Tabinda

    2011-01-01

    Dissection is being jeopardized in the modern medical education. It has unrelentingly faced the lashes of time and has been the scapegoat for numerous convenient curricula reforms and subjective biases. The cadaver is unparallel in establishing core knowledge among the medical community and it needs to be appreciated in a new light in the “cyber anatomy” realm of today. This article elucidates the medical and ethical validity of continuing human body dissection in medicine which outweighs all the prejudices associated with it. PMID:23908746

  11. Search Databases and Statistics: Pitfalls and Best Practices in Phosphoproteomics.

    PubMed

    Refsgaard, Jan C; Munk, Stephanie; Jensen, Lars J

    2016-01-01

    Advances in mass spectrometric instrumentation in the past 15 years have resulted in an explosion in the raw data yield from typical phosphoproteomics workflows. This poses the challenge of confidently identifying peptide sequences, localizing phosphosites to proteins and quantifying these from the vast amounts of raw data. This task is tackled by computational tools implementing algorithms that match the experimental data to databases, providing the user with lists for downstream analysis. Several platforms for such automated interpretation of mass spectrometric data have been developed, each having strengths and weaknesses that must be considered for the individual needs. These are reviewed in this chapter. Equally critical for generating highly confident output datasets is the application of sound statistical criteria to limit the inclusion of incorrect peptide identifications from database searches. Additionally, careful filtering and use of appropriate statistical tests on the output datasets affects the quality of all downstream analyses and interpretation of the data. Our considerations and general practices on these aspects of phosphoproteomics data processing are presented here. PMID:26584936

  12. A shotgun phosphoproteomics analysis of embryos in germinated maize seeds.

    PubMed

    Lu, Tian-Cong; Meng, Ling-Bo; Yang, Chuan-Ping; Liu, Gui-Feng; Liu, Guan-Jun; Ma, Wei; Wang, Bai-Chen

    2008-11-01

    To better understand the role that reversible protein phosphorylation plays in seed germination, we initiated a phosphoproteomic investigation of embryos of germinated maize seeds. A total of 776 proteins including 39 kinases, 16 phosphatases, and 33 phosphoproteins containing 36 precise in vivo phosphorylation sites were identified. All the phosphorylation sites identified, with the exception of the phosphorylation site on HSP22, have not been reported previously (Lund et al. in J Biol Chem, 276, 29924-29929, 2001). Assayed with QRT-PCR, the transcripts of ten kinase genes were found to be dramatically up-regulated during seed germination and those of four phosphatase genes were up-regulated after germination, which indicated that reversible protein phosphorylation occurred and complex regulating networks were activated during this period. At least one-third of these phosphoproteins are key components involved in biological processes which relate to seed germination, such as DNA repair, gene transcription, RNA splicing and protein translation, suggesting that protein phosphorylation plays an important role in seed germination. As far as we know, this is the first phosphoproteomic study on a monocot and it will lay a solid foundation for further study of the molecular mechanisms of seed germination and seedling development.

  13. Search Databases and Statistics: Pitfalls and Best Practices in Phosphoproteomics.

    PubMed

    Refsgaard, Jan C; Munk, Stephanie; Jensen, Lars J

    2016-01-01

    Advances in mass spectrometric instrumentation in the past 15 years have resulted in an explosion in the raw data yield from typical phosphoproteomics workflows. This poses the challenge of confidently identifying peptide sequences, localizing phosphosites to proteins and quantifying these from the vast amounts of raw data. This task is tackled by computational tools implementing algorithms that match the experimental data to databases, providing the user with lists for downstream analysis. Several platforms for such automated interpretation of mass spectrometric data have been developed, each having strengths and weaknesses that must be considered for the individual needs. These are reviewed in this chapter. Equally critical for generating highly confident output datasets is the application of sound statistical criteria to limit the inclusion of incorrect peptide identifications from database searches. Additionally, careful filtering and use of appropriate statistical tests on the output datasets affects the quality of all downstream analyses and interpretation of the data. Our considerations and general practices on these aspects of phosphoproteomics data processing are presented here.

  14. Two Dimensional Gel Electrophoresis-Based Plant Phosphoproteomics.

    PubMed

    Han, Chao; Yang, Pingfang

    2016-01-01

    Phosphorylation is one of the most important reversible protein modifications and is involved in regulating signal transduction, subcellular localization and enzyme activity of target proteins. Phosphorylation or dephosphorylation of proteins is directly reflected in changed ratios of phosphoprotein abundance and total protein abundance. Two-dimensional gel electrophoresis (2-DE)-based proteomics allow quantification of both total protein abundance by Coomassie Brilliant Blue (CBB) staining and phosphoprotein abundance by fluorescence-based staining. Pro-Q diamond phosphoprotein stain (Pro-Q DPS) can bind to the phosphate moiety of the phospho-amino acid directly, regardless of the nature of the phospho-amino acid. Phosphoproteins can thus be detected using proper excitation light, quantified using image analysis software and subsequently be subjected to analysis by mass spectrometry. Here, we describe a protein phosphorylation status analysis method combining both CBB and Pro-Q DPS staining based on 2-DE gel-based phosphoproteomics, which has been widely applied to plant phosphoproteomics studies.

  15. Spontaneous dissection of celiac trunk with concurrent splenic artery dissection

    PubMed Central

    Lee, Seo In; Kim, Jin Joo; Yang, Hyuk Jun; Lee, Keun

    2015-01-01

    Isolated spontaneous dissection of the celiac trunk is rarely diagnosed in acute abdominal pain. We present two cases of celiac trunk and splenic artery dissection with splenic infarction. Patients were successfully managed and stabilized by medical treatment. Isolated celiac trunk dissection can be fatal, therefore providers should be careful not to overlook this entity. PMID:27752606

  16. Dissecting Diversity Part II

    ERIC Educational Resources Information Center

    Matthews, Frank

    2005-01-01

    This article presents "Dissecting Diversity, Part II," the conclusion of a wide-ranging two-part roundtable discussion on diversity in higher education. The participants were as follows: Lezli Baskerville, J.D., President and CEO of the National Association for Equal Opportunity (NAFEO); Dr. Gerald E. Gipp, Executive Director of the American…

  17. Diagnosis of Intracranial Artery Dissection

    PubMed Central

    KANOTO, Masafumi; HOSOYA, Takaaki

    2016-01-01

    Cerebral arterial dissection is defined as a hematoma in the wall of a cervical or an intracranial artery. Cerebral arterial dissection causes arterial stenosis, occlusion, and aneurysm, resulting in acute infarction and hemorrhage. Image analysis by such methods as conventional angiography, computed tomography, magnetic resonance imaging, and so on plays an important role in diagnosing cerebral arterial dissection. In this study, we explore the methods and findings involved in the diagnosis of cerebral arterial dissection. PMID:27180630

  18. In-depth phosphoproteomic analysis of royal jelly derived from western and eastern honeybee species.

    PubMed

    Han, Bin; Fang, Yu; Feng, Mao; Lu, Xiaoshan; Huo, Xinmei; Meng, Lifeng; Wu, Bin; Li, Jianke

    2014-12-01

    The proteins in royal jelly (RJ) play a pivotal role in the nutrition, immune defense, and cast determination of honeybee larvae and have a wide range of pharmacological and health-promoting functions for humans as well. Although the importance of post-translational modifications (PTMs) in protein function is known, investigation of protein phosphorylation of RJ proteins is still very limited. To this end, two complementary phosphopeptide enrichment materials (Ti(4+)-IMAC and TiO2) and high-sensitivity mass spectrometry were applied to establish a detailed phosphoproteome map and to qualitatively and quantitatively compare the phosphoproteomes of RJ produced by Apis mellifera ligustica (Aml) and Apis cerana cerana (Acc). In total, 16 phosphoproteins carrying 67 phosphorylation sites were identified in RJ derived from western bees, and nine proteins phosphorylated on 71 sites were found in RJ produced by eastern honeybees. Of which, eight phosphorylated proteins were common to both RJ samples, and the same motif ([S-x-E]) was extracted, suggesting that the function of major RJ proteins as nutrients and immune agents is evolutionary preserved in both of these honeybee species. All eight overlapping phosphoproteins showed significantly higher abundance in Acc-RJ than in Aml-RJ, and the phosphorylation of Jelleine-II (an antimicrobial peptide, TPFKLSLHL) at S(6) in Acc-RJ had stronger antimicrobial properties than that at T(1) in Aml-RJ even though the overall antimicrobial activity of Jelleine-II was found to decrease after phosphorylation. The differences in phosphosites, peptide abundance, and antimicrobial activity of the phosphorylated RJ proteins indicate that the two major honeybee species employ distinct phosphorylation strategies that align with their different biological characteristics shaped by evolution. The phosphorylation of RJ proteins are potentially driven by the activity of extracellular serine/threonine protein kinase FAM20C-like protein (FAM20C

  19. Mechanisms of Soybean Roots' Tolerances to Salinity Revealed by Proteomic and Phosphoproteomic Comparisons Between Two Cultivars.

    PubMed

    Pi, Erxu; Qu, Liqun; Hu, Jianwen; Huang, Yingying; Qiu, Lijuan; Lu, Hongfei; Jiang, Bo; Liu, Cong; Peng, Tingting; Zhao, Ying; Wang, Huizhong; Tsai, Sau-Na; Ngai, Saiming; Du, Liqun

    2016-01-01

    Understanding molecular mechanisms underlying plant salinity tolerance provides valuable knowledgebase for effective crop improvement through genetic engineering. Current proteomic technologies, which support reliable and high-throughput analyses, have been broadly used for exploring sophisticated molecular networks in plants. In the current study, we compared phosphoproteomic and proteomic changes in roots of different soybean seedlings of a salt-tolerant cultivar (Wenfeng07) and a salt-sensitive cultivar (Union85140) induced by salt stress. The root samples of Wenfeng07 and Union85140 at three-trifoliate stage were collected at 0 h, 0.5 h, 1 h, 4 h, 12 h, 24 h, and 48 h after been treated with 150 mm NaCl. LC-MS/MS based phosphoproteomic analysis of these samples identified a total of 2692 phosphoproteins and 5509 phosphorylation sites. Of these, 2344 phosphoproteins containing 3744 phosphorylation sites were quantitatively analyzed. Our results showed that 1163 phosphorylation sites were differentially phosphorylated in the two compared cultivars. Among them, 10 MYB/MYB transcription factor like proteins were identified with fluctuating phosphorylation modifications at different time points, indicating that their crucial roles in regulating flavonol accumulation might be mediated by phosphorylated modifications. In addition, the protein expression profiles of these two cultivars were compared using LC MS/MS based shotgun proteomic analysis, and expression pattern of all the 89 differentially expressed proteins were independently confirmed by qRT-PCR. Interestingly, the enzymes involved in chalcone metabolic pathway exhibited positive correlations with salt tolerance. We confirmed the functional relevance of chalcone synthase, chalcone isomerase, and cytochrome P450 monooxygenase genes using soybean composites and Arabidopsis thaliana mutants, and found that their salt tolerance were positively regulated by chalcone synthase, but was negatively regulated by

  20. In-depth phosphoproteomic analysis of royal jelly derived from western and eastern honeybee species.

    PubMed

    Han, Bin; Fang, Yu; Feng, Mao; Lu, Xiaoshan; Huo, Xinmei; Meng, Lifeng; Wu, Bin; Li, Jianke

    2014-12-01

    The proteins in royal jelly (RJ) play a pivotal role in the nutrition, immune defense, and cast determination of honeybee larvae and have a wide range of pharmacological and health-promoting functions for humans as well. Although the importance of post-translational modifications (PTMs) in protein function is known, investigation of protein phosphorylation of RJ proteins is still very limited. To this end, two complementary phosphopeptide enrichment materials (Ti(4+)-IMAC and TiO2) and high-sensitivity mass spectrometry were applied to establish a detailed phosphoproteome map and to qualitatively and quantitatively compare the phosphoproteomes of RJ produced by Apis mellifera ligustica (Aml) and Apis cerana cerana (Acc). In total, 16 phosphoproteins carrying 67 phosphorylation sites were identified in RJ derived from western bees, and nine proteins phosphorylated on 71 sites were found in RJ produced by eastern honeybees. Of which, eight phosphorylated proteins were common to both RJ samples, and the same motif ([S-x-E]) was extracted, suggesting that the function of major RJ proteins as nutrients and immune agents is evolutionary preserved in both of these honeybee species. All eight overlapping phosphoproteins showed significantly higher abundance in Acc-RJ than in Aml-RJ, and the phosphorylation of Jelleine-II (an antimicrobial peptide, TPFKLSLHL) at S(6) in Acc-RJ had stronger antimicrobial properties than that at T(1) in Aml-RJ even though the overall antimicrobial activity of Jelleine-II was found to decrease after phosphorylation. The differences in phosphosites, peptide abundance, and antimicrobial activity of the phosphorylated RJ proteins indicate that the two major honeybee species employ distinct phosphorylation strategies that align with their different biological characteristics shaped by evolution. The phosphorylation of RJ proteins are potentially driven by the activity of extracellular serine/threonine protein kinase FAM20C-like protein (FAM20C

  1. Global phosphoproteomics of activated B cells using complementary metal ion functionalized soluble nanopolymers.

    PubMed

    Jayasundera, Keerthi B; Iliuk, Anton B; Nguyen, Andrew; Higgins, Renee; Geahlen, Robert L; Tao, W Andy

    2014-07-01

    Engagement of the B cell receptor for antigen (BCR) leads to immune responses through a cascade of intracellular signaling events. Most studies to date have focused on the BCR and protein tyrosine phosphorylation. Because spleen tyrosine kinase, Syk, is an upstream kinase in multiple BCR-regulated signaling pathways, it also affects many downstream events that are modulated through the phosphorylation of proteins on serine and threonine residues. Here, we report a novel phosphopeptide enrichment strategy and its application to a comprehensive quantitative phosphoproteomics analysis of Syk-dependent downstream signaling events in B cells, focusing on serine and threonine phosphorylation. Using a combination of the Syk inhibitor piceatannol, SILAC quantification, peptide fractionation, and complementary PolyMAC-Ti and PolyMAC-Zr enrichment techniques, we analyzed changes in BCR-stimulated protein phosphorylation that were dependent on the activity of Syk. We identified and quantified over 13,000 unique phosphopeptides, with a large percentage dependent on Syk activity in BCR-stimulated B cells. Our results not only confirmed many known functions of Syk, but more importantly, suggested many novel roles, including in the ubiquitin proteasome pathway, that warrant further exploration.

  2. Global Phosphoproteomics of Activated B Cells Using Complementary Metal Ion Functionalized Soluble Nanopolymers

    PubMed Central

    2015-01-01

    Engagement of the B cell receptor for antigen (BCR) leads to immune responses through a cascade of intracellular signaling events. Most studies to date have focused on the BCR and protein tyrosine phosphorylation. Because spleen tyrosine kinase, Syk, is an upstream kinase in multiple BCR-regulated signaling pathways, it also affects many downstream events that are modulated through the phosphorylation of proteins on serine and threonine residues. Here, we report a novel phosphopeptide enrichment strategy and its application to a comprehensive quantitative phosphoproteomics analysis of Syk-dependent downstream signaling events in B cells, focusing on serine and threonine phosphorylation. Using a combination of the Syk inhibitor piceatannol, SILAC quantification, peptide fractionation, and complementary PolyMAC-Ti and PolyMAC-Zr enrichment techniques, we analyzed changes in BCR-stimulated protein phosphorylation that were dependent on the activity of Syk. We identified and quantified over 13 000 unique phosphopeptides, with a large percentage dependent on Syk activity in BCR-stimulated B cells. Our results not only confirmed many known functions of Syk, but more importantly, suggested many novel roles, including in the ubiquitin proteasome pathway, that warrant further exploration. PMID:24905233

  3. Phosphoproteomics reveals malaria parasite Protein Kinase G as a signalling hub regulating egress and invasion

    PubMed Central

    Alam, Mahmood M.; Solyakov, Lev; Bottrill, Andrew R.; Flueck, Christian; Siddiqui, Faiza A.; Singh, Shailja; Mistry, Sharad; Viskaduraki, Maria; Lee, Kate; Hopp, Christine S.; Chitnis, Chetan E.; Doerig, Christian; Moon, Robert W.; Green, Judith L.; Holder, Anthony A.; Baker, David A.; Tobin, Andrew B.

    2015-01-01

    Our understanding of the key phosphorylation-dependent signalling pathways in the human malaria parasite, Plasmodium falciparum, remains rudimentary. Here we address this issue for the essential cGMP-dependent protein kinase, PfPKG. By employing chemical and genetic tools in combination with quantitative global phosphoproteomics, we identify the phosphorylation sites on 69 proteins that are direct or indirect cellular targets for PfPKG. These PfPKG targets include proteins involved in cell signalling, proteolysis, gene regulation, protein export and ion and protein transport, indicating that cGMP/PfPKG acts as a signalling hub that plays a central role in a number of core parasite processes. We also show that PfPKG activity is required for parasite invasion. This correlates with the finding that the calcium-dependent protein kinase, PfCDPK1, is phosphorylated by PfPKG, as are components of the actomyosin complex, providing mechanistic insight into the essential role of PfPKG in parasite egress and invasion. PMID:26149123

  4. Integrative Phosphoproteomics Links IL-23R Signaling with Metabolic Adaptation in Lymphocytes

    PubMed Central

    Lochmatter, Corinne; Fischer, Roman; Charles, Philip D.; Yu, Zhanru; Powrie, Fiona; Kessler, Benedikt M.

    2016-01-01

    Interleukin (IL)-23 mediated signal transduction represents a major molecular mechanism underlying the pathology of inflammatory bowel disease, Crohn’s disease and ulcerative colitis. In addition, emerging evidence supports the role of IL-23-driven Th17 cells in inflammation. Components of the IL-23 signaling pathway, such as IL-23R, JAK2 and STAT3, have been characterized, but elements unique to this network as compared to other interleukins have not been readily explored. In this study, we have undertaken an integrative phosphoproteomics approach to better characterise downstream signaling events. To this end, we performed and compared phosphopeptide and phosphoprotein enrichment methodologies after activation of T lymphocytes by IL-23. We demonstrate the complementary nature of the two phosphoenrichment approaches by maximizing the capture of phosphorylation events. A total of 8202 unique phosphopeptides, and 4317 unique proteins were identified, amongst which STAT3, PKM2, CDK6 and LASP-1 showed induction of specific phosphorylation not readily observed after IL-2 stimulation. Interestingly, quantitative analysis revealed predominant phosphorylation of pre-existing STAT3 nuclear subsets in addition to translocation of phosphorylated STAT3 within 30 min after IL-23 stimulation. After IL-23R activation, a small subset of PKM2 also translocates to the nucleus and may contribute to STAT3 phosphorylation, suggesting multiple cellular responses including metabolic adaptation. PMID:27080861

  5. Alternatives To Dissection. Second Edition.

    ERIC Educational Resources Information Center

    DeRosa, Bill, Ed.; Winiarskyj, Lesia, Ed.

    This packet attempts to provide educationally sound alternatives to dissection in the classroom, thereby making it possible for teachers to eliminate dissection from the curriculum. This packet can also be used by educators who include dissection in their curricula but consider it important to respect the expression of students' ethical, moral, or…

  6. Proximal aortic dissection (dissecting aortic aneurysm) in a mature ostrich.

    PubMed

    Ferreras, M C; González, J; Pérez, V; Reyes, L E; Gómez, N; Pérez, C; Corpa, J M; García-Marín, J F

    2001-01-01

    The gross and histopathologic lesions observed in a case of spontaneous proximal aortic dissection (dissecting aortic aneurysm) in a mature ostrich are reported. At necropsy, a dissecting intramural hematoma was seen in the proximal aorta, extended about 12 cm distally from the aortic valves. Histopathologic changes in aortic dissection included fragmentation and disruption of elastic laminae, presence of cystic extracellular spaces, and pooling of ground substance in the tunica media. Hepatic copper levels were measured, and the low concentration found suggested that a copper deficiency together with other risk factors such as the elevation of blood pressure may have been implicated in the development of the aortic dissection seen in this ostrich.

  7. Rapid Phosphoproteomic Effects of Abscisic Acid (ABA) on Wild-Type and ABA Receptor-Deficient A. thaliana Mutants*

    PubMed Central

    Minkoff, Benjamin B.; Stecker, Kelly E.; Sussman, Michael R.

    2015-01-01

    Abscisic acid (ABA)1 is a plant hormone that controls many aspects of plant growth, including seed germination, stomatal aperture size, and cellular drought response. ABA interacts with a unique family of 14 receptor proteins. This interaction leads to the activation of a family of protein kinases, SnRK2s, which in turn phosphorylate substrates involved in many cellular processes. The family of receptors appears functionally redundant. To observe a measurable phenotype, four of the fourteen receptors have to be mutated to create a multilocus loss-of-function quadruple receptor (QR) mutant, which is much less sensitive to ABA than wild-type (WT) plants. Given these phenotypes, we asked whether or not a difference in ABA response between the WT and QR backgrounds would manifest on a phosphorylation level as well. We tested WT and QR mutant ABA response using isotope-assisted quantitative phosphoproteomics to determine what ABA-induced phosphorylation changes occur in WT plants within 5 min of ABA treatment and how that phosphorylation pattern is altered in the QR mutant. We found multiple ABA-induced phosphorylation changes that occur within 5 min of treatment, including three SnRK2 autophosphorylation events and phosphorylation on SnRK2 substrates. The majority of robust ABA-dependent phosphorylation changes observed were partially diminished in the QR mutant, whereas many smaller ABA-dependent phosphorylation changes observed in the WT were not responsive to ABA in the mutant. A single phosphorylation event was increased in response to ABA treatment in both the WT and QR mutant. A portion of the discovery data was validated using selected reaction monitoring-based targeted measurements on a triple quadrupole mass spectrometer. These data suggest that different subsets of phosphorylation events depend upon different subsets of the ABA receptor family to occur. Altogether, these data expand our understanding of the model by which the family of ABA receptors directs

  8. Rapid Phosphoproteomic Effects of Abscisic Acid (ABA) on Wild-Type and ABA Receptor-Deficient A. thaliana Mutants.

    PubMed

    Minkoff, Benjamin B; Stecker, Kelly E; Sussman, Michael R

    2015-05-01

    Abscisic acid (ABA)¹ is a plant hormone that controls many aspects of plant growth, including seed germination, stomatal aperture size, and cellular drought response. ABA interacts with a unique family of 14 receptor proteins. This interaction leads to the activation of a family of protein kinases, SnRK2s, which in turn phosphorylate substrates involved in many cellular processes. The family of receptors appears functionally redundant. To observe a measurable phenotype, four of the fourteen receptors have to be mutated to create a multilocus loss-of-function quadruple receptor (QR) mutant, which is much less sensitive to ABA than wild-type (WT) plants. Given these phenotypes, we asked whether or not a difference in ABA response between the WT and QR backgrounds would manifest on a phosphorylation level as well. We tested WT and QR mutant ABA response using isotope-assisted quantitative phosphoproteomics to determine what ABA-induced phosphorylation changes occur in WT plants within 5 min of ABA treatment and how that phosphorylation pattern is altered in the QR mutant. We found multiple ABA-induced phosphorylation changes that occur within 5 min of treatment, including three SnRK2 autophosphorylation events and phosphorylation on SnRK2 substrates. The majority of robust ABA-dependent phosphorylation changes observed were partially diminished in the QR mutant, whereas many smaller ABA-dependent phosphorylation changes observed in the WT were not responsive to ABA in the mutant. A single phosphorylation event was increased in response to ABA treatment in both the WT and QR mutant. A portion of the discovery data was validated using selected reaction monitoring-based targeted measurements on a triple quadrupole mass spectrometer. These data suggest that different subsets of phosphorylation events depend upon different subsets of the ABA receptor family to occur. Altogether, these data expand our understanding of the model by which the family of ABA receptors directs

  9. Aortic dissection--an update.

    PubMed

    Mukherjee, Debabrata; Eagle, Kim A

    2005-06-01

    Acute aortic dissection is a medical emergency with high morbidity and mortality requiring emergent diagnosis and therapy. Rapid advances in noninvasive imaging technology have facilitated the early diagnosis of this condition and should be considered in the differential diagnosis of any patient with chest, back, or abdominal pain. Emergent surgery is the treatment for patients with type A dissection while optimal medical therapy is appropriate in patients with uncomplicated type B dissection. Adequate beta-blockade is the cornerstone of medical therapy. Patients who survive acute aortic dissection need long-term medical therapy with beta-blockers and statins and appropriate serial imaging follow-up. Future advances in this field include biomarkers in the early diagnosis of acute aortic dissection and presymptomatic diagnosis with genetic screening. Overall patients with aortic dissection are at high risk for an adverse outcome and need to be managed aggressively in hospital and long term with frequent follow-up. PMID:15973249

  10. Unraveling Kinase Activation Dynamics Using Kinase-Substrate Relationships from Temporal Large-Scale Phosphoproteomics Studies.

    PubMed

    Domanova, Westa; Krycer, James; Chaudhuri, Rima; Yang, Pengyi; Vafaee, Fatemeh; Fazakerley, Daniel; Humphrey, Sean; James, David; Kuncic, Zdenka

    2016-01-01

    In response to stimuli, biological processes are tightly controlled by dynamic cellular signaling mechanisms. Reversible protein phosphorylation occurs on rapid time-scales (milliseconds to seconds), making it an ideal carrier of these signals. Advances in mass spectrometry-based proteomics have led to the identification of many tens of thousands of phosphorylation sites, yet for the majority of these the kinase is unknown and the underlying network topology of signaling networks therefore remains obscured. Identifying kinase substrate relationships (KSRs) is therefore an important goal in cell signaling research. Existing consensus sequence motif based prediction algorithms do not consider the biological context of KSRs, and are therefore insensitive to many other mechanisms guiding kinase-substrate recognition in cellular contexts. Here, we use temporal information to identify biologically relevant KSRs from Large-scale In Vivo Experiments (KSR-LIVE) in a data-dependent and automated fashion. First, we used available phosphorylation databases to construct a repository of existing experimentally-predicted KSRs. For each kinase in this database, we used time-resolved phosphoproteomics data to examine how its substrates changed in phosphorylation over time. Although substrates for a particular kinase clustered together, they often exhibited a different temporal pattern to the phosphorylation of the kinase. Therefore, although phosphorylation regulates kinase activity, our findings imply that substrate phosphorylation likely serve as a better proxy for kinase activity than kinase phosphorylation. KSR-LIVE can thereby infer which kinases are regulated within a biological context. Moreover, KSR-LIVE can also be used to automatically generate positive training sets for the subsequent prediction of novel KSRs using machine learning approaches. We demonstrate that this approach can distinguish between Akt and Rps6kb1, two kinases that share the same linear consensus motif

  11. Unraveling Kinase Activation Dynamics Using Kinase-Substrate Relationships from Temporal Large-Scale Phosphoproteomics Studies

    PubMed Central

    Chaudhuri, Rima; Yang, Pengyi; Vafaee, Fatemeh; Fazakerley, Daniel; Humphrey, Sean; James, David; Kuncic, Zdenka

    2016-01-01

    In response to stimuli, biological processes are tightly controlled by dynamic cellular signaling mechanisms. Reversible protein phosphorylation occurs on rapid time-scales (milliseconds to seconds), making it an ideal carrier of these signals. Advances in mass spectrometry-based proteomics have led to the identification of many tens of thousands of phosphorylation sites, yet for the majority of these the kinase is unknown and the underlying network topology of signaling networks therefore remains obscured. Identifying kinase substrate relationships (KSRs) is therefore an important goal in cell signaling research. Existing consensus sequence motif based prediction algorithms do not consider the biological context of KSRs, and are therefore insensitive to many other mechanisms guiding kinase-substrate recognition in cellular contexts. Here, we use temporal information to identify biologically relevant KSRs from Large-scale In Vivo Experiments (KSR-LIVE) in a data-dependent and automated fashion. First, we used available phosphorylation databases to construct a repository of existing experimentally-predicted KSRs. For each kinase in this database, we used time-resolved phosphoproteomics data to examine how its substrates changed in phosphorylation over time. Although substrates for a particular kinase clustered together, they often exhibited a different temporal pattern to the phosphorylation of the kinase. Therefore, although phosphorylation regulates kinase activity, our findings imply that substrate phosphorylation likely serve as a better proxy for kinase activity than kinase phosphorylation. KSR-LIVE can thereby infer which kinases are regulated within a biological context. Moreover, KSR-LIVE can also be used to automatically generate positive training sets for the subsequent prediction of novel KSRs using machine learning approaches. We demonstrate that this approach can distinguish between Akt and Rps6kb1, two kinases that share the same linear consensus motif

  12. Intraoperative aortic dissection

    PubMed Central

    Singh, Ajmer; Mehta, Yatin

    2015-01-01

    Intraoperative aortic dissection is a rare but fatal complication of open heart surgery. By recognizing the population at risk and by using a gentle operative technique in such patients, the surgeon can usually avoid iatrogenic injury to the aorta. Intraoperative transesophageal echocardiography and epiaortic scanning are invaluable for prompt diagnosis and determination of the extent of the injury. Prevention lies in the strict control of blood pressure during cannulation/decannulation, construction of proximal anastomosis, or in avoiding manipulation of the aorta in high-risk patients. Immediate repair using interposition graft or Dacron patch graft is warranted to reduce the high mortality associated with this complication. PMID:26440240

  13. Pyruvate dehydrogenase alpha 1 as a target of omega-3 polyunsaturated fatty acids in human prostate cancer through a global phosphoproteomic analysis.

    PubMed

    Zhao, Heng; Pflug, Beth R; Lai, Xianyin; Wang, Mu

    2016-09-01

    Prostate cancer is one of the leading cancers in men. Taking dietary supplements, such as fish oil (FO), which is rich in n-3 polyunsaturated fatty acids (PUFAs), has been employed as a strategy to lower prostate cancer risk and control disease progression. In this study, we investigated the global phosphoproteomic changes induced by FO using a combination of phosphoprotein-enrichment strategy and high-resolution tandem mass spectrometry. We found that FO induces many more phosphorylation changes than oleic acid when they both are compared to control group. Quantitative comparison between untreated group and FO- or oleic acid-treated groups uncovered a number of important protein phosphorylation changes induced by n-3PUFAs. This phosphoproteomic discovery study and the follow-up Western Blot validation study elucidate that phosphorylation levels of the two regulatory serine residues in pyruvate dehydrogenase alpha 1 (PDHA1), serine-232 and serine-300, are significantly decreased upon FO treatment. As expected, increased pyruvate dehydrogenase activity was also observed. This study suggests that FO-induced phosphorylation changes in PDHA1 is more likely related to the glucose metabolism pathway, and n-3 PUFAs may have a role in controlling the balance between lipid and glucose oxidation. PMID:27357730

  14. Phosphoproteomic analysis of protein kinase C signaling in Saccharomyces cerevisiae reveals Slt2 mitogen-activated protein kinase (MAPK)-dependent phosphorylation of eisosome core components.

    PubMed

    Mascaraque, Victoria; Hernáez, María Luisa; Jiménez-Sánchez, María; Hansen, Rasmus; Gil, Concha; Martín, Humberto; Cid, Víctor J; Molina, María

    2013-03-01

    The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1-cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module.

  15. Early phosphoproteomic changes in the mouse spleen during deoxynivalenol-induced ribotoxic stress.

    PubMed

    Pan, Xiao; Whitten, Douglas A; Wu, Ming; Chan, Christina; Wilkerson, Curtis G; Pestka, James J

    2013-09-01

    The trichothecene mycotoxin deoxynivalenol (DON) targets the innate immune system and is of public health significance because of its frequent presence in human and animal food. DON-induced proinflammatory gene expression and apoptosis in the lymphoid tissue have been associated with a ribotoxic stress response (RSR) that involves rapid phosphorylation of mitogen-activated protein kinases (MAPKs). To better understand the relationship between protein phosphorylation and DON's immunotoxic effects, stable isotope dimethyl labeling-based proteomics in conjunction with titanium dioxide chromatography was employed to quantitatively profile the immediate (≤ 30min) phosphoproteome changes in the spleens of mice orally exposed to 5mg/kg body weight DON. A total of 90 phosphoproteins indicative of novel phosphorylation events were significantly modulated by DON. In addition to critical branches and scaffolds of MAPK signaling being affected, DON exposure also altered phosphorylation of proteins that mediate phosphatidylinositol 3-kinase/AKT pathways. Gene ontology analysis revealed that DON exposure affected biological processes such as cytoskeleton organization, regulation of apoptosis, and lymphocyte activation and development, which likely contribute to immune dysregulation associated with DON-induced RSR. Consistent with these findings, DON impacted phosphorylation of proteins within diverse immune cell populations, including monocytes, macrophages, T cells, B cells, dendritic cells, and mast cells. Fuzzy c-means clustering analysis further indicated that DON evoked several distinctive temporal profiles of regulated phosphopeptides. Overall, the findings from this investigation can serve as a template for future focused exploration and modeling of cellular responses associated with the immunotoxicity evoked by DON and other ribotoxins.

  16. Selection of an Appropriate Protein Extraction Method to Study the Phosphoproteome of Maize Photosynthetic Tissue

    PubMed Central

    Luís, Inês M.; Alexandre, Bruno M.; Oliveira, M. Margarida

    2016-01-01

    Often plant tissues are recalcitrant and, due to that, methods relying on protein precipitation, such as TCA/acetone precipitation and phenol extraction, are usually the methods of choice for protein extraction in plant proteomic studies. However, the addition of precipitation steps to protein extraction methods may negatively impact protein recovery, due to problems associated with protein re-solubilization. Moreover, we show that when working with non-recalcitrant plant tissues, such as young maize leaves, protein extraction methods with precipitation steps compromise the maintenance of some labile post-translational modifications (PTMs), such as phosphorylation. Therefore, a critical issue when studying PTMs in plant proteins is to ensure that the protein extraction method is the most appropriate, both at qualitative and quantitative levels. In this work, we compared five methods for protein extraction of the C4-photosynthesis related proteins, in the tip of fully expanded third-leaves. These included: TCA/Acetone Precipitation; Phenol Extraction; TCA/Acetone Precipitation followed by Phenol Extraction; direct extraction in Lysis Buffer (a urea-based buffer); and direct extraction in Lysis Buffer followed by Cleanup with a commercial kit. Protein extraction in Lysis Buffer performed better in comparison to the other methods. It gave one of the highest protein yields, good coverage of the extracted proteome and phosphoproteome, high reproducibility, and little protein degradation. This was also the easiest and fastest method, warranting minimal sample handling. We also show that this method is adequate for the successful extraction of key enzymes of the C4-photosynthetic metabolism, such as PEPC, PPDK, PEPCK, and NADP-ME. This was confirmed by MALDI-TOF/TOF MS analysis of excised spots of 2DE analyses of the extracted protein pools. Staining for phosphorylated proteins in 2DE revealed the presence of several phosphorylated isoforms of PEPC, PPDK, and PEPCK. PMID

  17. DNA Replication Stress Phosphoproteome Profiles Reveal Novel Functional Phosphorylation Sites on Xrs2 in Saccharomyces cerevisiae.

    PubMed

    Huang, Dongqing; Piening, Brian D; Kennedy, Jacob J; Lin, Chenwei; Jones-Weinert, Corey W; Yan, Ping; Paulovich, Amanda G

    2016-05-01

    In response to replication stress, a phospho-signaling cascade is activated and required for coordination of DNA repair and replication of damaged templates (intra-S-phase checkpoint) . How phospho-signaling coordinates the DNA replication stress response is largely unknown. We employed state-of-the-art liquid chromatography tandem-mass spectrometry (LC-MS/MS) approaches to generate high-coverage and quantitative proteomic and phospho-proteomic profiles during replication stress in yeast, induced by continuous exposure to the DNA alkylating agent methyl methanesulfonate (MMS) . We identified 32,057 unique peptides representing the products of 4296 genes and 22,061 unique phosphopeptides representing the products of 3183 genes. A total of 542 phosphopeptides (mapping to 339 genes) demonstrated an abundance change of greater than or equal to twofold in response to MMS. The screen enabled detection of nearly all of the proteins known to be involved in the DNA damage response, as well as many novel MMS-induced phosphorylations. We assessed the functional importance of a subset of key phosphosites by engineering a panel of phosphosite mutants in which an amino acid substitution prevents phosphorylation. In total, we successfully mutated 15 MMS-responsive phosphorylation sites in seven representative genes including APN1 (base excision repair); CTF4 and TOF1 (checkpoint and sister-chromatid cohesion); MPH1 (resolution of homologous recombination intermediates); RAD50 and XRS2 (MRX complex); and RAD18 (PRR). All of these phosphorylation site mutants exhibited MMS sensitivity, indicating an important role in protecting cells from DNA damage. In particular, we identified MMS-induced phosphorylation sites on Xrs2 that are required for MMS resistance in the absence of the MRX activator, Sae2, and that affect telomere maintenance.

  18. DNA Replication Stress Phosphoproteome Profiles Reveal Novel Functional Phosphorylation Sites on Xrs2 in Saccharomyces cerevisiae.

    PubMed

    Huang, Dongqing; Piening, Brian D; Kennedy, Jacob J; Lin, Chenwei; Jones-Weinert, Corey W; Yan, Ping; Paulovich, Amanda G

    2016-05-01

    In response to replication stress, a phospho-signaling cascade is activated and required for coordination of DNA repair and replication of damaged templates (intra-S-phase checkpoint) . How phospho-signaling coordinates the DNA replication stress response is largely unknown. We employed state-of-the-art liquid chromatography tandem-mass spectrometry (LC-MS/MS) approaches to generate high-coverage and quantitative proteomic and phospho-proteomic profiles during replication stress in yeast, induced by continuous exposure to the DNA alkylating agent methyl methanesulfonate (MMS) . We identified 32,057 unique peptides representing the products of 4296 genes and 22,061 unique phosphopeptides representing the products of 3183 genes. A total of 542 phosphopeptides (mapping to 339 genes) demonstrated an abundance change of greater than or equal to twofold in response to MMS. The screen enabled detection of nearly all of the proteins known to be involved in the DNA damage response, as well as many novel MMS-induced phosphorylations. We assessed the functional importance of a subset of key phosphosites by engineering a panel of phosphosite mutants in which an amino acid substitution prevents phosphorylation. In total, we successfully mutated 15 MMS-responsive phosphorylation sites in seven representative genes including APN1 (base excision repair); CTF4 and TOF1 (checkpoint and sister-chromatid cohesion); MPH1 (resolution of homologous recombination intermediates); RAD50 and XRS2 (MRX complex); and RAD18 (PRR). All of these phosphorylation site mutants exhibited MMS sensitivity, indicating an important role in protecting cells from DNA damage. In particular, we identified MMS-induced phosphorylation sites on Xrs2 that are required for MMS resistance in the absence of the MRX activator, Sae2, and that affect telomere maintenance. PMID:27017623

  19. Early Phosphoproteomic Changes in the Mouse Spleen During Deoxynivalenol-Induced Ribotoxic Stress

    PubMed Central

    Pestka, James J.

    2013-01-01

    The trichothecene mycotoxin deoxynivalenol (DON) targets the innate immune system and is of public health significance because of its frequent presence in human and animal food. DON-induced proinflammatory gene expression and apoptosis in the lymphoid tissue have been associated with a ribotoxic stress response (RSR) that involves rapid phosphorylation of mitogen-activated protein kinases (MAPKs). To better understand the relationship between protein phosphorylation and DON’s immunotoxic effects, stable isotope dimethyl labeling–based proteomics in conjunction with titanium dioxide chromatography was employed to quantitatively profile the immediate (≤ 30min) phosphoproteome changes in the spleens of mice orally exposed to 5mg/kg body weight DON. A total of 90 phosphoproteins indicative of novel phosphorylation events were significantly modulated by DON. In addition to critical branches and scaffolds of MAPK signaling being affected, DON exposure also altered phosphorylation of proteins that mediate phosphatidylinositol 3-kinase/AKT pathways. Gene ontology analysis revealed that DON exposure affected biological processes such as cytoskeleton organization, regulation of apoptosis, and lymphocyte activation and development, which likely contribute to immune dysregulation associated with DON-induced RSR. Consistent with these findings, DON impacted phosphorylation of proteins within diverse immune cell populations, including monocytes, macrophages, T cells, B cells, dendritic cells, and mast cells. Fuzzy c-means clustering analysis further indicated that DON evoked several distinctive temporal profiles of regulated phosphopeptides. Overall, the findings from this investigation can serve as a template for future focused exploration and modeling of cellular responses associated with the immunotoxicity evoked by DON and other ribotoxins. PMID:23811945

  20. "Dissection" of a Hair Dryer

    ERIC Educational Resources Information Center

    Eisenstein, Stan; Simpson, Jeff

    2008-01-01

    The electrical design of the common hair dryer is based almost entirely on relatively simple principles learned in introductory physics classes. Just as biology students dissect a frog to see the principles of anatomy in action, physics students can "dissect" a hair dryer to see how principles of electricity are used in a real system. They can…

  1. Tinea capitis mimicking dissecting cellulitis.

    PubMed

    Torok, Rachel D; Bellet, Jane S

    2013-01-01

    Tinea capitis is a common disease of childhood that typically follows one of several clinical patterns. Our patient and several previously reported cases demonstrate the existence of a dissecting cellulitis-like presentation of tinea capitis. This variant should be recognized to prevent misdiagnosis of dissecting cellulitis and allow proper treatment to prevent scarring alopecia. PMID:24134312

  2. Tinea capitis mimicking dissecting cellulitis.

    PubMed

    Torok, Rachel D; Bellet, Jane S

    2013-01-01

    Tinea capitis is a common disease of childhood that typically follows one of several clinical patterns. Our patient and several previously reported cases demonstrate the existence of a dissecting cellulitis-like presentation of tinea capitis. This variant should be recognized to prevent misdiagnosis of dissecting cellulitis and allow proper treatment to prevent scarring alopecia.

  3. Recent advances in enrichment and separation strategies for mass spectrometry-based phosphoproteomics

    PubMed Central

    Yang, Chenxi; Zhong, Xuefei; Li, Lingjun

    2016-01-01

    Due to the significance of protein phosphorylation in various biological processes and signaling events, new analytical techniques for enhanced phosphoproteomics have been rapidly introduced in recent years. The combinatorial use of the phospho-specific enrichment techniques and prefractionation methods prior to MS analysis enables comprehensive profiling of the phosphoproteome and facilitates deciphering the critical roles that phosphorylation plays in signaling pathways in various biological systems. This review places special emphasis on the recent five-year (2009–2013) advances for enrichment and separation techniques that have been utilized for phosphopeptides prior to MS analysis. PMID:24687451

  4. [Stent Grafting for Aortic Dissection].

    PubMed

    Uchida, Naomichi

    2016-07-01

    The purpose of stent graft for aortic dissection is to terminate antegrade blood flow into the false lumen through primary entry. Early intervention for primary entry makes excellent aortic remodeling and emergent stent grafting for complicated acute type B aortic dissection is supported as a class I. On the other hand stent grafting for chronic aortic dissection is controversial. Early stent grafting is considered with in 6 months after on-set if the diameter of the descending aorta is more than 40 mm. Additional interventions for residual false lumen on the downstream aorta are still required. Stent graft for re-entry, candy-plug technique, and double stenting, other effective re-interventions were reported. Best treatment on the basis of each anatomical and physical characteristics should be selected in each institution. Frozen elephant trunk is alternative procedure for aortic dissection without the need to take account of proximal anatomical limitation and effective for acute type A aortic dissection. PMID:27440026

  5. Automatic Dissection Of Plantlets

    NASA Astrophysics Data System (ADS)

    Batchelor, B. G.; Harris, I. P.; Marchant, J. A.; Tillett, R. D.

    1989-03-01

    Micropropagation is a technique used in horticulture for generating a monoclonal colony of plants. A tiny plantlet is cut into several parts, each of which is then replanted. At the moment, the cutting is performed manually. Automating this task would have significant economic benefits. A robot designed to dissect plants would need to be equipped with intelligent visual sensing. This article is concerned with the image acquisition and processing techniques which such a machine might use. A program, which can calculate where to cut a plant with an "open" structure, is presented. This is expressed in the ProVision language, which is described in another article presented at this conference. (Article 1002-65)

  6. Spontaneous Coronary Artery Dissection.

    PubMed

    Tweet, Marysia S; Gulati, Rajiv; Hayes, Sharonne N

    2016-07-01

    Spontaneous coronary artery dissection is an important etiology of nonatherosclerotic acute coronary syndrome, myocardial infarction, and sudden death. Innovations in the catheterization laboratory including optical coherence tomography and intravascular ultrasound have enhanced the ability to visualize intimal disruption and intramural hematoma associated with SCAD. Formerly considered "rare," these technological advances and heightened awareness suggest that SCAD is more prevalent than prior estimates. SCAD is associated with female sex, young age, extreme emotional stress, or extreme exertion, pregnancy, and fibromuscular dysplasia. The clinical characteristics and management strategies of SCAD patients are different than for atherosclerotic heart disease and deserve specific consideration. This review will highlight recent discoveries about SCAD as well as describe current efforts to elucidate remaining gaps in knowledge.

  7. Spontaneous Coronary Artery Dissection.

    PubMed

    Tweet, Marysia S; Gulati, Rajiv; Hayes, Sharonne N

    2016-07-01

    Spontaneous coronary artery dissection is an important etiology of nonatherosclerotic acute coronary syndrome, myocardial infarction, and sudden death. Innovations in the catheterization laboratory including optical coherence tomography and intravascular ultrasound have enhanced the ability to visualize intimal disruption and intramural hematoma associated with SCAD. Formerly considered "rare," these technological advances and heightened awareness suggest that SCAD is more prevalent than prior estimates. SCAD is associated with female sex, young age, extreme emotional stress, or extreme exertion, pregnancy, and fibromuscular dysplasia. The clinical characteristics and management strategies of SCAD patients are different than for atherosclerotic heart disease and deserve specific consideration. This review will highlight recent discoveries about SCAD as well as describe current efforts to elucidate remaining gaps in knowledge. PMID:27216840

  8. How Safe is High-Power Ultrasonic Dissection?

    PubMed Central

    Emam, Tarek A.; Cuschieri, Alfred

    2003-01-01

    Objective To evaluate the safety of ultrasonic dissection. Summary Background Data High-power ultrasonic dissection is in widespread use for both open and laparoscopic operations and is generally perceived to carry a low risk of collateral damage, but there is no published evidence for this. Methods Under controlled experimental conditions, ultrasonic dissections were performed in pigs using Ultracision (Ethicon) or Autosonix (Tyco/USSC) at the three power settings (3, 4, and 5) in random fashion to mobilize the cardia and fundus, bile duct, hepatic artery, portal vein, aorta from the inferior vena cava, renal vessels, colon, and ureters. The dissections (open and laparoscopic) were carried out on pigs at each power setting with each device. Thermal mapping of the tissues during dissection was performed with an infrared thermal camera and associated software. The animals were killed at the end of each experiment and specimens were harvested for quantitative histology. Results Extreme and equivalent temperature gradients were generated by ultrasonic dissection with both systems. Heat production was directly proportional to the power setting and the activation time. The core body temperature of the animals after completion of the laparoscopic dissections rose by an average of 2.3°C. The zone around the jaws that exceeded 60°C with continuous ultrasonic dissection for 10 to 15 seconds at level 5 measured 25.3 and 25.7 mm for Ultracision and Autosonix, respectively. At this power setting and an activation time of 15 seconds, the temperature 1.0 cm away from the tips of the instrument exceeded 140°C. Although there was no discernible macroscopic damage, these thermal changes were accompanied by significant histologic injury that extended to the media of large vessels and caused partial- to full-thickness mural damage of the cardia, ureter, and bile duct. Collateral damage was absent or insignificant after dissections at power level 3 with both systems and an

  9. [Dissection techniques in liver surgery].

    PubMed

    Rau, H G; Schauer, R; Pickelmann, S; Beyer, B C; Angele, M K; Zimmermann, A; Meimarakis, G; Heizmann, O; Schildberg, F W

    2001-02-01

    The first liver resection was performed in 1888. Since then a wide variety of dissection techniques have been introduced. The blunt dissection was replaced by novel methods, i.e. the CUSA technique and the Jet Cutter for major liver resections. These methods represent selective dissection techniques; whereas non-selective methods include the scalpel, scissors, linear stapling cutter, high-frequency coagulation, and the laser technique. The aim of this review article is the comparison of the different resection techniques in liver surgery, focussing on blood loss and resection time. PMID:11253668

  10. Computational Fluid Dynamics Analysis of Thoracic Aortic Dissection

    NASA Astrophysics Data System (ADS)

    Tang, Yik; Fan, Yi; Cheng, Stephen; Chow, Kwok

    2011-11-01

    Thoracic Aortic Dissection (TAD) is a cardiovascular disease with high mortality. An aortic dissection is formed when blood infiltrates the layers of the vascular wall, and a new artificial channel, the false lumen, is created. The expansion of the blood vessel due to the weakened wall enhances the risk of rupture. Computational fluid dynamics analysis is performed to study the hemodynamics of this pathological condition. Both idealized geometry and realistic patient configurations from computed tomography (CT) images are investigated. Physiological boundary conditions from in vivo measurements are employed. Flow configuration and biomechanical forces are studied. Quantitative analysis allows clinicians to assess the risk of rupture in making decision regarding surgical intervention.

  11. Wrangling Phosphoproteomic Data to Elucidate Cancer Signaling Pathways

    PubMed Central

    Grimes, Mark L.; Lee, Wan-Jui; van der Maaten, Laurens; Shannon, Paul

    2013-01-01

    The interpretation of biological data sets is essential for generating hypotheses that guide research, yet modern methods of global analysis challenge our ability to discern meaningful patterns and then convey results in a way that can be easily appreciated. Proteomic data is especially challenging because mass spectrometry detectors often miss peptides in complex samples, resulting in sparsely populated data sets. Using the R programming language and techniques from the field of pattern recognition, we have devised methods to resolve and evaluate clusters of proteins related by their pattern of expression in different samples in proteomic data sets. We examined tyrosine phosphoproteomic data from lung cancer samples. We calculated dissimilarities between the proteins based on Pearson or Spearman correlations and on Euclidean distances, whilst dealing with large amounts of missing data. The dissimilarities were then used as feature vectors in clustering and visualization algorithms. The quality of the clusterings and visualizations were evaluated internally based on the primary data and externally based on gene ontology and protein interaction networks. The results show that t-distributed stochastic neighbor embedding (t-SNE) followed by minimum spanning tree methods groups sparse proteomic data into meaningful clusters more effectively than other methods such as k-means and classical multidimensional scaling. Furthermore, our results show that using a combination of Spearman correlation and Euclidean distance as a dissimilarity representation increases the resolution of clusters. Our analyses show that many clusters contain one or more tyrosine kinases and include known effectors as well as proteins with no known interactions. Visualizing these clusters as networks elucidated previously unknown tyrosine kinase signal transduction pathways that drive cancer. Our approach can be applied to other data types, and can be easily adopted because open source software

  12. Comprehensive Analysis of in Vivo Phosphoproteome of Mouse Liver Microsomes.

    PubMed

    Kwon, Oh Kwang; Sim, JuHee; Kim, Sun Ju; Sung, Eunji; Kim, Jin Young; Jeong, Tae Cheon; Lee, Sangkyu

    2015-12-01

    Protein phosphorylation at serine, threonine, and tyrosine residues are some of the most widespread reversible post-translational modifications. Microsomes are vesicle-like bodies, not ordinarily present within living cells, which form from pieces of the endoplasmic reticulum (ER), plasma membrane, mitochondria, or Golgi apparatus of broken eukaryotic cells. Here we investigated the total phosphoproteome of mouse liver microsomes (MLMs) using TiO2 enrichment of phosphopeptides coupled to on-line 2D-LC-MS/MS. In total, 699 phosphorylation sites in 527 proteins were identified in MLMs. When compared with the current phosphoSitePlus database, 155 novel phosphoproteins were identified in MLM. The distributions of phosphosites were 89.4, 8.0, and 2.6% for phosphoserine, phosphotheronine, and phosphotyrosine, respectively. By Motif-X analysis, eight Ser motifs and one Thr motif were found, and five acidic, two basophilic-, and two proline-directed motifs were assigned. The potential functions of phosphoproteins in MLM were assigned by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. In GO annotation, phosphorylated microsomal proteins were involved in mRNA processing, mRNA metabolic processes, and RNA splicing. In the KEGG pathway analysis, phosphorylated microsomal proteins were highly enriched in ribosome protein processing in ER and ribosomes and in RNA transport. Furthermore, we determined that 52 and 23 phosphoproteins were potential substrates of cAMP-dependent protein kinase A and casein kinase II, respectively, many of which are 40S/60S ribosomal proteins. Overall, our results provide an overview of features of protein phosphorylation in MLMs that should be a valuable resource for the future understanding of protein synthesis or translation involving phosphorylation. PMID:26487105

  13. Experience with parametric binary dissection

    NASA Technical Reports Server (NTRS)

    Bokhari, Shahid H.

    1993-01-01

    Parametric Binary Dissection (PBD) is a new algorithm that can be used for partitioning graphs embedded in 2- or 3-dimensional space. It partitions explicitly on the basis of nodes + (lambda)x(edges cut), where lambda is the ratio of time to communicate over an edge to the time to compute at a node. The new algorithm is faster than the original binary dissection algorithm and attempts to obtain better partitions than the older algorithm, which only takes nodes into account. The performance of parametric dissection with plain binary dissection on 3 large unstructured 3-d meshes obtained from computational fluid dynamics and on 2 random graphs were compared. It was showm that the new algorithm can usually yield partitions that are substantially superior, but that its performance is heavily dependent on the input data.

  14. Decreased expression of fibulin-4 in aortic wall of aortic dissection.

    PubMed

    Huawei, P; Qian, C; Chuan, T; Lei, L; Laing, W; Wenlong, X; Wenzhi, L

    2014-02-01

    In this research, we will examine the expression of Fibulin-4 in aortic wall to find out its role in aortic dissection development. The samples of aortic wall were obtained from 10 patients operated for acute ascending aortic dissection and five patients for chronic ascending aortic dissection. Another 15 pieces of samples from patients who had coronary artery bypass were as controls. The aortic samples were stained with aldehyde magenta dyeing to evaluate the arrangement of elastic fibers. The Fibulin-4 protein and mRNA expression were both determined by Western blot and realtime quantitative polymerase chain reaction. Compared with the control group, both in acute and chronic ascending aortic dissection, elastic fiber fragments increased and the expression of fibulin-4 protein significantly decreased (P= 0.045 < 0.05). The level of fibulin-4 mRNA decreased in acute ascending aortic dissection (P= 0.034 < 0.05), while it increased in chronic ascending aortic dissection (P=0.004 < 0.05). The increased amounts of elastic fiber fragments were negatively correlated with the expression of fibulin-4 mRNA in acute ascending aortic dissection. In conclusion, in aortic wall of ascending aortic dissection, the expression of fibulin-4 protein decreased and the expression of fibulin-4 mRNA was abnormal. Fibulin-4 may play an important role in the pathogenesis of aortic dissection.

  15. Phosphoproteome Analysis Reveals the Molecular Mechanisms Underlying Deoxynivalenol-Induced Intestinal Toxicity in IPEC-J2 Cells.

    PubMed

    Zhang, Zhi-Qi; Wang, Song-Bo; Wang, Rui-Guo; Zhang, Wei; Wang, Pei-Long; Su, Xiao-Ou

    2016-01-01

    Deoxynivalenol (DON) is a widespread trichothecene mycotoxin that commonly contaminates cereal crops and has various toxic effects in animals and humans. DON primarily targets the gastrointestinal tract, the first barrier against ingested food contaminants. In this study, an isobaric tag for relative and absolute quantitation (iTRAQ)-based phosphoproteomic approach was employed to elucidate the molecular mechanisms underlying DON-mediated intestinal toxicity in porcine epithelial cells (IPEC-J2) exposed to 20 μM DON for 60 min. There were 4153 unique phosphopeptides, representing 389 phosphorylation sites, detected in 1821 phosphoproteins. We found that 289 phosphopeptides corresponding to 255 phosphoproteins were differentially phosphorylated in response to DON. Comprehensive Gene Ontology (GO) analysis combined with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment revealed that, in addition to previously well-characterized mitogen-activated protein kinase (MAPK) signaling, DON exposure altered phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and Janus kinase/signal transducer, and activator of transcription (JAK/STAT) pathways. These pathways are involved in a wide range of biological processes, including apoptosis, the intestinal barrier, intestinal inflammation, and the intestinal absorption of glucose. DON-induced changes are likely to contribute to the intestinal dysfunction. Overall, identification of relevant signaling pathways yielded new insights into the molecular mechanisms underlying DON-induced intestinal toxicity, and might help in the development of improved mechanism-based risk assessments in animals and humans. PMID:27669298

  16. Phosphoproteomic profiling identifies focal adhesion kinase as a mediator of docetaxel resistance in castrate-resistant prostate cancer.

    PubMed

    Lee, Brian Y; Hochgräfe, Falko; Lin, Hui-Ming; Castillo, Lesley; Wu, Jianmin; Raftery, Mark J; Martin Shreeve, S; Horvath, Lisa G; Daly, Roger J

    2014-01-01

    Docetaxel remains the standard-of-care for men diagnosed with metastatic castrate-resistant prostate cancer (CRPC). However, only approximately 50% of patients benefit from treatment and all develop docetaxel-resistant disease. Here, we characterize global perturbations in tyrosine kinase signaling associated with docetaxel resistance and thereby develop a potential therapeutic strategy to reverse this phenotype. Using quantitative mass spectrometry-based phosphoproteomics, we identified that metastatic docetaxel-resistant prostate cancer cell lines (DU145-Rx and PC3-Rx) exhibit increased phosphorylation of focal adhesion kinase (FAK) on Y397 and Y576, in comparison with parental controls (DU145 and PC3, respectively). Bioinformatic analyses identified perturbations in pathways regulating focal adhesions and the actin cytoskeleton and in protein-protein interaction networks related to these pathways in docetaxel-resistant cells. Treatment with the FAK tyrosine kinase inhibitor (TKI) PF-00562271 reduced FAK phosphorylation in the resistant cells, but did not affect cell viability or Akt phosphorylation. Docetaxel administration reduced FAK and Akt phosphorylation, whereas cotreatment with PF-00562271 and docetaxel resulted in an additive attenuation of FAK and Akt phosphorylation and overcame the chemoresistant phenotype. The enhanced efficacy of cotreatment was due to increased autophagic cell death, rather than apoptosis. These data strongly support that enhanced FAK activation mediates chemoresistance in CRPC, and identify a potential clinical niche for FAK TKIs, where coadministration with docetaxel may be used in patients with CRPC to overcome chemoresistance. PMID:24194567

  17. Phosphoproteomic profiling of tumor tissues identifies HSP27 Ser82 phosphorylation as a robust marker of early ischemia

    PubMed Central

    Zahari, Muhammad Saddiq; Wu, Xinyan; Pinto, Sneha M.; Nirujogi, Raja Sekhar; Kim, Min-Sik; Fetics, Barry; Philip, Mathew; Barnes, Sheri R.; Godfrey, Beverly; Gabrielson, Edward; Nevo, Erez; Pandey, Akhilesh

    2015-01-01

    Delays between tissue collection and tissue fixation result in ischemia and ischemia-associated changes in protein phosphorylation levels, which can misguide the examination of signaling pathway status. To identify a biomarker that serves as a reliable indicator of ischemic changes that tumor tissues undergo, we subjected harvested xenograft tumors to room temperature for 0, 2, 10 and 30 minutes before freezing in liquid nitrogen. Multiplex TMT-labeling was conducted to achieve precise quantitation, followed by TiO2 phosphopeptide enrichment and high resolution mass spectrometry profiling. LC-MS/MS analyses revealed phosphorylation level changes of a number of phosphosites in the ischemic samples. The phosphorylation of one of these sites, S82 of the heat shock protein 27 kDa (HSP27), was especially abundant and consistently upregulated in tissues with delays in freezing as short as 2 minutes. In order to eliminate effects of ischemia, we employed a novel cryogenic biopsy device which begins freezing tissues in situ before they are excised. Using this device, we showed that the upregulation of phosphorylation of S82 on HSP27 was abrogated. We thus demonstrate that our cryogenic biopsy device can eliminate ischemia-induced phosphoproteome alterations, and measurements of S82 on HSP27 can be used as a robust marker of ischemia in tissues. PMID:26329039

  18. Phosphoproteome Analysis Reveals the Molecular Mechanisms Underlying Deoxynivalenol-Induced Intestinal Toxicity in IPEC-J2 Cells

    PubMed Central

    Zhang, Zhi-Qi; Wang, Song-Bo; Wang, Rui-Guo; Zhang, Wei; Wang, Pei-Long; Su, Xiao-Ou

    2016-01-01

    Deoxynivalenol (DON) is a widespread trichothecene mycotoxin that commonly contaminates cereal crops and has various toxic effects in animals and humans. DON primarily targets the gastrointestinal tract, the first barrier against ingested food contaminants. In this study, an isobaric tag for relative and absolute quantitation (iTRAQ)-based phosphoproteomic approach was employed to elucidate the molecular mechanisms underlying DON-mediated intestinal toxicity in porcine epithelial cells (IPEC-J2) exposed to 20 μM DON for 60 min. There were 4153 unique phosphopeptides, representing 389 phosphorylation sites, detected in 1821 phosphoproteins. We found that 289 phosphopeptides corresponding to 255 phosphoproteins were differentially phosphorylated in response to DON. Comprehensive Gene Ontology (GO) analysis combined with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment revealed that, in addition to previously well-characterized mitogen-activated protein kinase (MAPK) signaling, DON exposure altered phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and Janus kinase/signal transducer, and activator of transcription (JAK/STAT) pathways. These pathways are involved in a wide range of biological processes, including apoptosis, the intestinal barrier, intestinal inflammation, and the intestinal absorption of glucose. DON-induced changes are likely to contribute to the intestinal dysfunction. Overall, identification of relevant signaling pathways yielded new insights into the molecular mechanisms underlying DON-induced intestinal toxicity, and might help in the development of improved mechanism-based risk assessments in animals and humans. PMID:27669298

  19. Phosphoproteome Analysis Reveals the Molecular Mechanisms Underlying Deoxynivalenol-Induced Intestinal Toxicity in IPEC-J2 Cells.

    PubMed

    Zhang, Zhi-Qi; Wang, Song-Bo; Wang, Rui-Guo; Zhang, Wei; Wang, Pei-Long; Su, Xiao-Ou

    2016-09-22

    Deoxynivalenol (DON) is a widespread trichothecene mycotoxin that commonly contaminates cereal crops and has various toxic effects in animals and humans. DON primarily targets the gastrointestinal tract, the first barrier against ingested food contaminants. In this study, an isobaric tag for relative and absolute quantitation (iTRAQ)-based phosphoproteomic approach was employed to elucidate the molecular mechanisms underlying DON-mediated intestinal toxicity in porcine epithelial cells (IPEC-J2) exposed to 20 μM DON for 60 min. There were 4153 unique phosphopeptides, representing 389 phosphorylation sites, detected in 1821 phosphoproteins. We found that 289 phosphopeptides corresponding to 255 phosphoproteins were differentially phosphorylated in response to DON. Comprehensive Gene Ontology (GO) analysis combined with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment revealed that, in addition to previously well-characterized mitogen-activated protein kinase (MAPK) signaling, DON exposure altered phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and Janus kinase/signal transducer, and activator of transcription (JAK/STAT) pathways. These pathways are involved in a wide range of biological processes, including apoptosis, the intestinal barrier, intestinal inflammation, and the intestinal absorption of glucose. DON-induced changes are likely to contribute to the intestinal dysfunction. Overall, identification of relevant signaling pathways yielded new insights into the molecular mechanisms underlying DON-induced intestinal toxicity, and might help in the development of improved mechanism-based risk assessments in animals and humans.

  20. Phosphoproteome analysis demonstrates the potential role of THRAP3 phosphorylation in androgen-independent prostate cancer cell growth.

    PubMed

    Ino, Yoko; Arakawa, Noriaki; Ishiguro, Hitoshi; Uemura, Hiroji; Kubota, Yoshinobu; Hirano, Hisashi; Toda, Tosifusa

    2016-04-01

    Elucidating the androgen-independent growth mechanism is critical for developing effective treatment strategies to combat androgen-independent prostate cancer. We performed a comparative phosphoproteome analysis using a prostate cancer cell line, LNCaP, and an LNCaP-derived androgen-independent cell line, LNCaP-AI, to identify phosphoproteins involved in this mechanism. We performed quantitative comparisons of the phosphopeptide levels in tryptic digests of protein extracts from these cell lines using MS. We found that the levels of 69 phosphopeptides in 66 proteins significantly differed between LNCaP and LNCaP-AI. In particular, we focused on thyroid hormone receptor associated protein 3 (THRAP3), which is a known transcriptional coactivator of the androgen receptor. The phosphorylation level of THRAP3 was significantly lower at S248 and S253 in LNCaP-AI cells. Furthermore, pull-down assays showed that 32 proteins uniquely bound to the nonphosphorylatable mutant form of THRAP3, whereas 31 other proteins uniquely bound to the phosphorylation-mimic form. Many of the differentially interacting proteins were identified as being involved with RNA splicing and processing. These results suggest that the phosphorylation state of THRAP3 at S248 and S253 might be involved in the mechanism of androgen-independent prostate cancer cell growth by changing the interaction partners.

  1. Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1.

    PubMed

    Lai, Yu-Chiang; Kondapalli, Chandana; Lehneck, Ronny; Procter, James B; Dill, Brian D; Woodroof, Helen I; Gourlay, Robert; Peggie, Mark; Macartney, Thomas J; Corti, Olga; Corvol, Jean-Christophe; Campbell, David G; Itzen, Aymelt; Trost, Matthias; Muqit, Miratul Mk

    2015-11-12

    Mutations in the PTEN-induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson's disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser(65)) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1-dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub-family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser(111)) in response to PINK1 activation. Using phospho-specific antibodies raised against Ser(111) of each of the Rabs, we demonstrate that Rab Ser(111) phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient-derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser(111) phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser(111) phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser(65). We further show mechanistically that phosphorylation at Ser(111) significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser(111) may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase-mediated signalling may represent a major mechanism

  2. Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1.

    PubMed

    Lai, Yu-Chiang; Kondapalli, Chandana; Lehneck, Ronny; Procter, James B; Dill, Brian D; Woodroof, Helen I; Gourlay, Robert; Peggie, Mark; Macartney, Thomas J; Corti, Olga; Corvol, Jean-Christophe; Campbell, David G; Itzen, Aymelt; Trost, Matthias; Muqit, Miratul Mk

    2015-11-12

    Mutations in the PTEN-induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson's disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser(65)) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1-dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub-family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser(111)) in response to PINK1 activation. Using phospho-specific antibodies raised against Ser(111) of each of the Rabs, we demonstrate that Rab Ser(111) phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient-derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser(111) phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser(111) phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser(65). We further show mechanistically that phosphorylation at Ser(111) significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser(111) may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase-mediated signalling may represent a major mechanism

  3. Online, Absolute Quantitation of Propranolol from Spatially Distinct 20- and 40-μm Dissections of Brain, Liver, and Kidney Thin Tissue Sections by Laser Microdissection-Liquid Vortex Capture-Mass Spectrometry.

    PubMed

    Cahill, John F; Kertesz, Vilmos; Weiskittel, Taylor M; Vavrek, Marissa; Freddo, Carol; Van Berkel, Gary J

    2016-06-01

    Spatial resolved quantitation of chemical species in thin tissue sections by mass spectrometric methods has been constrained by the need for matrix-matched standards or other arduous calibration protocols and procedures to mitigate matrix effects (e.g., spatially varying ionization suppression). Reported here is the use of laser "cut and drop" sampling with a laser microdissection-liquid vortex capture electrospray ionization tandem mass spectrometry (LMD-LVC/ESI-MS/MS) system for online and absolute quantitation of propranolol in mouse brain, kidney, and liver thin tissue sections of mice administered with the drug at a 7.5 mg/kg dose, intravenously. In this procedure either 20 μm × 20 μm or 40 μm × 40 μm tissue microdissections were cut and dropped into the flowing solvent of the capture probe. During transport to the ESI source drug related material was completely extracted from the tissue into the solvent, which contained a known concentration of propranolol-d7 as an internal standard. This allowed absolute quantitation to be achieved with an external calibration curve generated from standards containing the same fixed concentration of propranolol-d7 and varied concentrations of propranolol. Average propranolol concentrations determined with the laser "cut and drop" sampling method closely agreed with concentration values obtained from 2.3 mm diameter tissue punches from serial sections that were extracted and quantified by HPLC/ESI-MS/MS measurements. In addition, the relative abundance of hydroxypropranolol glucuronide metabolites were recorded and found to be consistent with previous findings. PMID:27214103

  4. Online, absolute quantitation of propranolol from spatially distinct 20-μm and 40-μm dissections of brain, liver, and kidney thin tissue sections by laser microdissection – liquid vortex capture – mass spectrometry

    DOE PAGESBeta

    Kertesz, Vilmos; Vavrek, Marissa; Freddo, Carol; Van Berkel, Gary J.; Cahill, John F.; Weiskittel, Taylor M.

    2016-05-23

    Here, spatial resolved quantitation of chemical species in thin tissue sections by mass spectrometric methods has been constrained by the need for matrix-matched standards or other arduous calibration protocols and procedures to mitigate matrix effects (e.g., spatially varying ionization suppression). Reported here is the use of laser cut and drop sampling with a laser microdissection-liquid vortex capture electrospray ionization tandem mass spectrometry (LMD-LVC/ESI-MS/MS) system for online and absolute quantitation of propranolol in mouse brain, kidney, and liver thin tissue sections of mice administered with the drug at a 7.5 mg/kg dose, intravenously. In this procedure either 20 μm x 20more » μm or 40 μm x 40 μm tissue microdissections were cut and dropped into the flowing solvent of the capture probe. During transport to the ESI source drug related material was completely extracted from the tissue into the solvent, which contained a known concentration of propranolol-d7 as an internal standard. This allowed absolute quantitation to be achieved with an external calibration curve generated from standards containing the same fixed concentration of propranolold-d7 and varied concentrations of propranolol. Average propranolol concentrations determined with the laser cut and drop sampling method closely agreed with concentration values obtained from 2.3 mm diameter tissue punches from serial sections that were extracted and quantified by HPLC/ESI-MS/MS measurements. In addition, the relative abundance of hydroxypropranolol glucuronide metabolites were recorded and found to be consistent with previous findings.« less

  5. Spontaneous internal carotid artery dissection.

    PubMed

    Khimenko, L P; Esham, H R; Ahmed, W

    2000-10-01

    Once considered uncommon, spontaneous dissection of the carotid artery is an increasingly recognized cause of stroke, headache, cranial nerve palsy, or ophthalmologic events, especially in young adults. Even in the presence of existing signs and symptoms, the diagnosis can be missed by experienced physicians of all specialties. We report a case of spontaneous internal carotid artery dissection in a 38-year-old woman with a cortical stroke and visual disturbances as initial symptoms. The diagnosis was confirmed by magnetic resonance imaging/angiography and by angiography. Prompt anticoagulation was instituted, and the patient had complete resolution of symptoms. Cervicocephalic arterial dissection should be included in the differential diagnosis of the causes of cerebrovascular events.

  6. ``Dissection'' of a Hair Dryer

    NASA Astrophysics Data System (ADS)

    Eisenstein, Stan; Simpson, Jeff

    2008-12-01

    The electrical design of the common hair dryer is based almost entirely on relatively simple principles learned in introductory physics classes. Just as biology students dissect a frog to see the principles of anatomy in action, physics students can "dissect" a hair dryer to see how principles of electricity are used in a real system. They can discover how engineers solve problems such as how to vary between low and high heat and fan speed by simply moving the position of a single switch. Principles of alternating versus direct current, series and parallel circuits, electrical safety, voltage dividing, ac rectification, power, and measurement of resistance and continuity all come in to play.

  7. [Pregnancy and coronary artery dissection].

    PubMed

    Martínez-Quintana, Efrén; Rodríguez-González, Fayna

    2015-01-01

    Acute myocardial infarction during pregnancy is associated with high maternal and fetal mortality. Coronary atherosclerosis is the most common cause due to an increase in the age of the patients and the association with cardiovascular risk factors such as smoking, hypertension, diabetes mellitus, preeclampsia, and the existence of family history of coronary disease. However, thrombosis, coronary dissection or coronary vasospasms are other causes that may justify it. We report the case of a 33 weeks pregnant first-time mother, without cardiovascular risk factors, who presented an acute coronary event in the context of atherosclerotic disease and coronary dissection after percutaneous coronary intervention.

  8. [Pregnancy and coronary artery dissection].

    PubMed

    Martínez-Quintana, Efrén; Rodríguez-González, Fayna

    2015-01-01

    Acute myocardial infarction during pregnancy is associated with high maternal and fetal mortality. Coronary atherosclerosis is the most common cause due to an increase in the age of the patients and the association with cardiovascular risk factors such as smoking, hypertension, diabetes mellitus, preeclampsia, and the existence of family history of coronary disease. However, thrombosis, coronary dissection or coronary vasospasms are other causes that may justify it. We report the case of a 33 weeks pregnant first-time mother, without cardiovascular risk factors, who presented an acute coronary event in the context of atherosclerotic disease and coronary dissection after percutaneous coronary intervention. PMID:25795261

  9. Quantitative phosphoproteomics unveils temporal dynamics of thrombin signaling in human endothelial cells

    PubMed Central

    van den Biggelaar, Maartje; Hernández-Fernaud, Juan Ramon; van den Eshof, Bart L.; Neilson, Lisa J.; Meijer, Alexander B.; Mertens, Koen

    2014-01-01

    Thrombin is the key serine protease of the coagulation cascade and a potent trigger of protease-activated receptor 1 (PAR1)-mediated platelet aggregation. In recent years, PAR1 has become an appealing target for anticoagulant therapies. However, the inhibitors that have been developed so far increase bleeding risk in patients, likely because they interfere with endogenous PAR1 signaling in the endothelium. Because of its complexity, thrombin-induced signaling in endothelial cells has remained incompletely understood. Here, we have combined stable isotope amino acids in cell culture, affinity-based phosphopeptide enrichment, and high-resolution mass spectrometry and performed a time-resolved analysis of the thrombin-induced signaling in human primary endothelial cells. We identified 2224 thrombin-regulated phosphorylation sites, the majority of which have not been previously related to thrombin. Those sites were localized on proteins that are novel to thrombin signaling, but also on well-known players such as PAR1, Rho-associated kinase 2, phospholipase C, and proteins related to actin cytoskeleton, cell-cell junctions, and Weibel-Palade body release. Our study provides a unique resource of phosphoproteins and phosphorylation sites that may generate novel insights into an intimate understanding of thrombin-mediated PAR signaling and the development of improved PAR1 antagonists that affect platelet but not endothelial cell function. PMID:24501219

  10. Quantitative phosphoproteomics unveils temporal dynamics of thrombin signaling in human endothelial cells.

    PubMed

    van den Biggelaar, Maartje; Hernández-Fernaud, Juan Ramon; van den Eshof, Bart L; Neilson, Lisa J; Meijer, Alexander B; Mertens, Koen; Zanivan, Sara

    2014-03-20

    Thrombin is the key serine protease of the coagulation cascade and a potent trigger of protease-activated receptor 1 (PAR1)-mediated platelet aggregation. In recent years, PAR1 has become an appealing target for anticoagulant therapies. However, the inhibitors that have been developed so far increase bleeding risk in patients, likely because they interfere with endogenous PAR1 signaling in the endothelium. Because of its complexity, thrombin-induced signaling in endothelial cells has remained incompletely understood. Here, we have combined stable isotope amino acids in cell culture, affinity-based phosphopeptide enrichment, and high-resolution mass spectrometry and performed a time-resolved analysis of the thrombin-induced signaling in human primary endothelial cells. We identified 2224 thrombin-regulated phosphorylation sites, the majority of which have not been previously related to thrombin. Those sites were localized on proteins that are novel to thrombin signaling, but also on well-known players such as PAR1, Rho-associated kinase 2, phospholipase C, and proteins related to actin cytoskeleton, cell-cell junctions, and Weibel-Palade body release. Our study provides a unique resource of phosphoproteins and phosphorylation sites that may generate novel insights into an intimate understanding of thrombin-mediated PAR signaling and the development of improved PAR1 antagonists that affect platelet but not endothelial cell function. PMID:24501219

  11. [Progress and application of phosphoproteomics in the proteomics of liver pathological and physiological state].

    PubMed

    Yi, Tailong; Tian, Miaomiao; Yang, Xiaoming; Xu, Ping

    2014-07-01

    The phosphorylation is one of most common protein post-translational modifications. The protein phosphorylation plays important roles in the life through the reversible process of phosphorylation and dephosphorylation by kinases and phosphatases. Systematical analysis of the phosphorylation state of proteins would greatly help to reveal the mystery of the life. Recently, with the development of mass spectrometer, bioinformatics sortwares and enrichment methods of phosphopeptides, phosphorylation stduy of orgnism proteins by mass spectrometer has become mature gradually. Liver is one of the most important metabolic and immune organs. In-depth study of protein phosphorylation in liver is of great importance to reveal its function. And booming phosphoproteomics has been applied into the study of liver, which has deepened the knowledge of molecular mechnism of its physiology and pathology states. Here, we review the recent progress on the research and development of phosphoproteomics and their application in liver proteomics study.

  12. Phosphoproteome of the cyanobacterium Synechocystis sp. PCC 6803 and its dynamics during nitrogen starvation

    PubMed Central

    Spät, Philipp; Maček, Boris; Forchhammer, Karl

    2015-01-01

    Cyanobacteria have shaped the earth's biosphere as the first oxygenic photoautotrophs and still play an important role in many ecosystems. The ability to adapt to changing environmental conditions is an essential characteristic in order to ensure survival. To this end, numerous studies have shown that bacteria use protein post-translational modifications such as Ser/Thr/Tyr phosphorylation in cell signaling, adaptation, and regulation. Nevertheless, our knowledge of cyanobacterial phosphoproteomes and their dynamic response to environmental stimuli is relatively limited. In this study, we applied gel-free methods and high accuracy mass spectrometry toward the detection of Ser/Thr/Tyr phosphorylation events in the model cyanobacterium Synechocystis sp. PCC 6803. We could identify over 300 phosphorylation events in cultures grown on nitrate as exclusive nitrogen source. Chemical dimethylation labeling was applied to investigate proteome and phosphoproteome dynamics during nitrogen starvation. Our dataset describes the most comprehensive (phospho)proteome of Synechocystis to date, identifying 2382 proteins and 183 phosphorylation events and quantifying 2111 proteins and 148 phosphorylation events during nitrogen starvation. Global protein phosphorylation levels were increased in response to nitrogen depletion after 24 h. Among the proteins with increased phosphorylation, the PII signaling protein showed the highest fold-change, serving as positive control. Other proteins with increased phosphorylation levels comprised functions in photosynthesis and in carbon and nitrogen metabolism. This study reveals dynamics of Synechocystis phosphoproteome in response to environmental stimuli and suggests an important role of protein Ser/Thr/Tyr phosphorylation in fundamental mechanisms of homeostatic control in cyanobacteria. PMID:25873915

  13. Phosphoproteome of the cyanobacterium Synechocystis sp. PCC 6803 and its dynamics during nitrogen starvation.

    PubMed

    Spät, Philipp; Maček, Boris; Forchhammer, Karl

    2015-01-01

    Cyanobacteria have shaped the earth's biosphere as the first oxygenic photoautotrophs and still play an important role in many ecosystems. The ability to adapt to changing environmental conditions is an essential characteristic in order to ensure survival. To this end, numerous studies have shown that bacteria use protein post-translational modifications such as Ser/Thr/Tyr phosphorylation in cell signaling, adaptation, and regulation. Nevertheless, our knowledge of cyanobacterial phosphoproteomes and their dynamic response to environmental stimuli is relatively limited. In this study, we applied gel-free methods and high accuracy mass spectrometry toward the detection of Ser/Thr/Tyr phosphorylation events in the model cyanobacterium Synechocystis sp. PCC 6803. We could identify over 300 phosphorylation events in cultures grown on nitrate as exclusive nitrogen source. Chemical dimethylation labeling was applied to investigate proteome and phosphoproteome dynamics during nitrogen starvation. Our dataset describes the most comprehensive (phospho)proteome of Synechocystis to date, identifying 2382 proteins and 183 phosphorylation events and quantifying 2111 proteins and 148 phosphorylation events during nitrogen starvation. Global protein phosphorylation levels were increased in response to nitrogen depletion after 24 h. Among the proteins with increased phosphorylation, the PII signaling protein showed the highest fold-change, serving as positive control. Other proteins with increased phosphorylation levels comprised functions in photosynthesis and in carbon and nitrogen metabolism. This study reveals dynamics of Synechocystis phosphoproteome in response to environmental stimuli and suggests an important role of protein Ser/Thr/Tyr phosphorylation in fundamental mechanisms of homeostatic control in cyanobacteria. PMID:25873915

  14. Searching for novel Cdk5 substrates in brain by comparative phosphoproteomics of wild type and Cdk5-/- mice.

    PubMed

    Contreras-Vallejos, Erick; Utreras, Elías; Bórquez, Daniel A; Prochazkova, Michaela; Terse, Anita; Jaffe, Howard; Toledo, Andrea; Arruti, Cristina; Pant, Harish C; Kulkarni, Ashok B; González-Billault, Christian

    2014-01-01

    Protein phosphorylation is the most common post-translational modification that regulates several pivotal functions in cells. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase which is mostly active in the nervous system. It regulates several biological processes such as neuronal migration, cytoskeletal dynamics, axonal guidance and synaptic plasticity among others. In search for novel substrates of Cdk5 in the brain we performed quantitative phosphoproteomics analysis, isolating phosphoproteins from whole brain derived from E18.5 Cdk5+/+ and Cdk5-/- embryos, using an Immobilized Metal-Ion Affinity Chromatography (IMAC), which specifically binds to phosphorylated proteins. The isolated phosphoproteins were eluted and isotopically labeled for relative and absolute quantitation (iTRAQ) and mass spectrometry identification. We found 40 proteins that showed decreased phosphorylation at Cdk5-/- brains. In addition, out of these 40 hypophosphorylated proteins we characterized two proteins, :MARCKS (Myristoylated Alanine-Rich protein Kinase C substrate) and Grin1 (G protein regulated inducer of neurite outgrowth 1). MARCKS is known to be phosphorylated by Cdk5 in chick neural cells while Grin1 has not been reported to be phosphorylated by Cdk5. When these proteins were overexpressed in N2A neuroblastoma cell line along with p35, serine phosphorylation in their Cdk5 motifs was found to be increased. In contrast, treatments with roscovitine, the Cdk5 inhibitor, resulted in an opposite effect on serine phosphorylation in N2A cells and primary hippocampal neurons transfected with MARCKS. In summary, the results presented here identify Grin 1 as novel Cdk5 substrate and confirm previously identified MARCKS as a a bona fide Cdk5 substrate.

  15. Searching for novel Cdk5 substrates in brain by comparative phosphoproteomics of wild type and Cdk5-/- mice.

    PubMed

    Contreras-Vallejos, Erick; Utreras, Elías; Bórquez, Daniel A; Prochazkova, Michaela; Terse, Anita; Jaffe, Howard; Toledo, Andrea; Arruti, Cristina; Pant, Harish C; Kulkarni, Ashok B; González-Billault, Christian

    2014-01-01

    Protein phosphorylation is the most common post-translational modification that regulates several pivotal functions in cells. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase which is mostly active in the nervous system. It regulates several biological processes such as neuronal migration, cytoskeletal dynamics, axonal guidance and synaptic plasticity among others. In search for novel substrates of Cdk5 in the brain we performed quantitative phosphoproteomics analysis, isolating phosphoproteins from whole brain derived from E18.5 Cdk5+/+ and Cdk5-/- embryos, using an Immobilized Metal-Ion Affinity Chromatography (IMAC), which specifically binds to phosphorylated proteins. The isolated phosphoproteins were eluted and isotopically labeled for relative and absolute quantitation (iTRAQ) and mass spectrometry identification. We found 40 proteins that showed decreased phosphorylation at Cdk5-/- brains. In addition, out of these 40 hypophosphorylated proteins we characterized two proteins, :MARCKS (Myristoylated Alanine-Rich protein Kinase C substrate) and Grin1 (G protein regulated inducer of neurite outgrowth 1). MARCKS is known to be phosphorylated by Cdk5 in chick neural cells while Grin1 has not been reported to be phosphorylated by Cdk5. When these proteins were overexpressed in N2A neuroblastoma cell line along with p35, serine phosphorylation in their Cdk5 motifs was found to be increased. In contrast, treatments with roscovitine, the Cdk5 inhibitor, resulted in an opposite effect on serine phosphorylation in N2A cells and primary hippocampal neurons transfected with MARCKS. In summary, the results presented here identify Grin 1 as novel Cdk5 substrate and confirm previously identified MARCKS as a a bona fide Cdk5 substrate. PMID:24658276

  16. Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates.

    PubMed

    Xue, Liang; Wang, Wen-Horng; Iliuk, Anton; Hu, Lianghai; Galan, Jacob A; Yu, Shuai; Hans, Michael; Geahlen, Robert L; Tao, W Andy

    2012-04-10

    Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity. PMID:22451900

  17. Dissect Your Squid and Eat It Too!

    ERIC Educational Resources Information Center

    McGinnis, Patricia

    2001-01-01

    Introduces a science lab activity in which students dissect fresh squids in groups of four and observe the anatomy. Parent volunteers cook the squid mantle for kids to taste. Includes directions for squid dissection. (YDS)

  18. Dissection & Science Fairs. [Information Packet.

    ERIC Educational Resources Information Center

    National Anti-Vivisection Society, Chicago, IL.

    This collection of pamphlets and articles reprinted from other National Anti-Vivisection Society (NAVS) publications was compiled to address the issues of classroom laboratory dissection and the use of animals in science fair projects. Three of the pamphlets contained in this packet are student handbooks designed to help students of elementary,…

  19. Chronic Type A Aortic Dissection

    PubMed Central

    Hynes, Conor F.; Greenberg, Michael D.; Sarin, Shawn; Trachiotis, Gregory D.

    2016-01-01

    Stanford Type A aortic dissection is a rapidly progressing disease process that is often fatal without emergent surgical repair. A small proportion of Type A dissections go undiagnosed in the acute phase and are found upon delayed presentation of symptoms or incidentally. These chronic lesions may have a distinct natural history that may have a better prognosis and could potentially be managed differently then those presenting acutely. The method of repair depends on location and extent of the false lumen, as well as involvement of critical structures and branch arteries. Surgical repair techniques similar to those employed for acute dissection management are currently first-line therapy for chronic cases that involve the aortic valve, sinuses of Valsalva, coronary arteries, and supra-aortic branch arteries. In patients with high-risk for surgery, endovascular repairs have been successful, and active development of delivery systems and grafts will continue to enhance outcomes. We present two cases of chronic Type A aortic dissection and review the current literature.

  20. Animal Rights Activism Threatens Dissection.

    ERIC Educational Resources Information Center

    Holden, Constance

    1990-01-01

    Discussed is the movement against the use of dissections in science laboratories. Examples of protests across the United States are included. Compared is the plight of using animals in a biology classroom and the demise of the teaching of evolution in some areas. (KR)

  1. Cutting Edge Controversy: The Politics of Animal Dissection and Responses to Student Objection

    NASA Astrophysics Data System (ADS)

    Oakley, Jan

    2011-12-01

    This mixed methods study investigated the experiences and perspectives of former Ontario high school students and current Ontario science and biology teachers toward animal dissection, objection to dissection, and choice policies that grant students the right to opt out of dissection and use an alternative instead. Data was collected via a student questionnaire (n=311), a teacher questionnaire (n=153), and interviews with eight students and nine teachers. Quantitative and qualitative data analyses and reporting techniques were employed within a humane education and critical pedagogy framework to explore the experiences and perspectives of both groups.

  2. Morphological Variation in Leaf Dissection of Rheum palmatum Complex (Polygonaceae)

    PubMed Central

    Wang, Xu-Mei; Hou, Xiao-Qi; Zhang, Yu-Qu; Li, Yan

    2014-01-01

    Aims Rheum palmatum complex comprises all taxa within section Palmata in the genus Rheum, including R. officinale, R. palmatum, R. tanguticum, R. tanguticum var. liupanshanense and R. laciniatum. The identification of the taxa in section Palmata is based primarily on the degree of leaf blade dissection and the shape of the lobes; however, difficulties in species identification may arise from their significant variation. The aim of this study is to analyze the patterns of variation in leaf blade characteristics within and among populations through population-based sampling covering the entire distribution range of R. palmatum complex. Methods Samples were taken from 2340 leaves from 780 individuals and 44 populations representing the four species, and the degree of leaf blade dissection and the shape of the lobe were measured to yield a set of quantitative data. Furthermore, those data were statistically analyzed. Important Findings The statistical analysis showed that the degree of leaf blade dissection is continuous from lobed to parted, and the shape of the lobe is also continuous from broadly triangular to lanceolate both within and between populations. We suggested that taxa in section Palmata should be considered as one species. Based on the research on the R. palmatum complex, we considered that the quantitative characteristics were greatly influenced by the environment. Therefore, it is not reliable to delimitate the species according to the continuously quantitative vegetative characteristics. PMID:25349989

  3. LoQAtE—Localization and Quantitation ATlas of the yeast proteomE. A new tool for multiparametric dissection of single-protein behavior in response to biological perturbations in yeast

    PubMed Central

    Breker, Michal; Gymrek, Melissa; Moldavski, Ofer; Schuldiner, Maya

    2014-01-01

    Living organisms change their proteome dramatically to sustain a stable internal milieu in fluctuating environments. To study the dynamics of proteins during stress, we measured the localization and abundance of the Saccharomyces cerevisiae proteome under various growth conditions and genetic backgrounds using the GFP collection. We created a database (DB) called ‘LoQAtE’ (Localizaiton and Quantitation Atlas of the yeast proteomE), available online at http://www.weizmann.ac.il/molgen/loqate/, to provide easy access to these data. Using LoQAtE DB, users can get a profile of changes for proteins of interest as well as querying advanced intersections by either abundance changes, primary localization or localization shifts over the tested conditions. Currently, the DB hosts information on 5330 yeast proteins under three external perturbations (DTT, H2O2 and nitrogen starvation) and two genetic mutations [in the chaperonin containing TCP1 (CCT) complex and in the proteasome]. Additional conditions will be uploaded regularly. The data demonstrate hundreds of localization and abundance changes, many of which were not detected at the level of mRNA. LoQAtE is designed to allow easy navigation for non-experts in high-content microscopy and data are available for download. These data should open up new perspectives on the significant role of proteins while combating external and internal fluctuations. PMID:24150937

  4. A Dissecting Competition for Medical Students

    ERIC Educational Resources Information Center

    Samalia, Latika; Stringer, Mark D.

    2012-01-01

    After repeated requests from medical students for more cadaver dissection opportunities, a voluntary dissecting "competition" was initiated for the third year medical students in 2006. This has been held annually on five occasions since, offering up to 30 dissection stations and accommodating an average of 53 students (range 40-66) per year,…

  5. Reflections on dissection: leave no student behind.

    PubMed

    Skandalakis, John E

    2008-01-01

    Dissection is being reduced--and even removed--from the medical curriculum in Australia, New Zealand, the United Kingdom, Ireland, and the United States. Dissection's contribution to the curriculum is too important to be diminished. To understand the human body, students must dissect. To avoid anatomical complications, future surgeons need the knowledge they can gain from dissection. Cadavers reveal the uniqueness of each body and the body's strength and fragility, which cannot be learned from books or computers. Cadavers offer surgical skill-building opportunities and confrontation with death. For all its strengths, however, dissection alone does not teach everything the student needs to know. Other educational tools (books, CT and MRI, animation of developmental processes) successfully fill in gaps of knowledge. Surgeons and educators must recognize the threat that decreased dissection poses to our students and patients. They must take steps to support dissection in the medical curriculum or, if it has disappeared, to bring it back.

  6. Dissecting cellulitis of the scalp.

    PubMed

    Mundi, Jyoti P; Marmon, Shoshana; Fischer, Max; Kamino, Hideko; Patel, Rishi; Shapiro, Jerry

    2012-12-01

    Dissecting cellulitis of the scalp is a chronic, relapsing, inflammatory disease of the scalp that results in scarring alopecia. We present a case of a 32-year-old man with recalcitrant disease who is now responding to treatment with isotretinoin. The pathogenesis, clinical presentation, disease associations, and histopathological findings are reviewed. Treatment can be challenging. The literature on medical and surgical therapeutic options is reviewed. PMID:23286798

  7. Combined Quantification of the Global Proteome, Phosphoproteome, and Proteolytic Cleavage to Characterize Altered Platelet Functions in the Human Scott Syndrome*

    PubMed Central

    Solari, Fiorella A.; Mattheij, Nadine J.A.; Burkhart, Julia M.; Swieringa, Frauke; Collins, Peter W.; Cosemans, Judith M.E.M.; Sickmann, Albert; Heemskerk, Johan W.M.; Zahedi, René P.

    2016-01-01

    The Scott syndrome is a very rare and likely underdiagnosed bleeding disorder associated with mutations in the gene encoding anoctamin-6. Platelets from Scott patients are impaired in various Ca2+-dependent responses, including phosphatidylserine exposure, integrin closure, intracellular protein cleavage, and cytoskeleton-dependent morphological changes. Given the central role of anoctamin-6 in the platelet procoagulant response, we used quantitative proteomics to understand the underlying molecular mechanisms and the complex phenotypic changes in Scott platelets compared with control platelets. Therefore, we applied an iTRAQ-based multi-pronged strategy to quantify changes in (1) the global proteome, (2) the phosphoproteome, and (3) proteolytic events between resting and stimulated Scott and control platelets. Our data indicate a limited number of proteins with decreased (70) or increased (64) expression in Scott platelets, among those we confirmed the absence of anoctamin-6 and the strong up-regulation of aquaporin-1 by parallel reaction monitoring. The quantification of 1566 phosphopeptides revealed major differences between Scott and control platelets after stimulation with thrombin/convulxin or ionomycin. In Scott platelets, phosphorylation levels of proteins regulating cytoskeletal or signaling events were increased. Finally, we quantified 1596 N-terminal peptides in activated Scott and control platelets, 180 of which we identified as calpain-regulated, whereas a distinct set of 23 neo-N termini was caspase-regulated. In Scott platelets, calpain-induced cleavage of cytoskeleton-linked and signaling proteins was downregulated, in accordance with an increased phosphorylation state. Thus, multipronged proteomic profiling of Scott platelets provides detailed insight into their protection against detrimental Ca2+-dependent changes that are normally associated with phosphatidylserine exposure. PMID:27535140

  8. Genetic dissection of acetic acid tolerance in Saccharomyces cerevisiae.

    PubMed

    Geng, Peng; Xiao, Yin; Hu, Yun; Sun, Haiye; Xue, Wei; Zhang, Liang; Shi, Gui-Yang

    2016-09-01

    Dissection of the hereditary architecture underlying Saccharomyces cerevisiae tolerance to acetic acid is essential for ethanol fermentation. In this work, a genomics approach was used to dissect hereditary variations in acetic acid tolerance between two phenotypically different strains. A total of 160 segregants derived from these two strains were obtained. Phenotypic analysis indicated that the acetic acid tolerance displayed a normal distribution in these segregants, and suggested that the acetic acid tolerant traits were controlled by multiple quantitative trait loci (QTLs). Thus, 220 SSR markers covering the whole genome were used to detect QTLs of acetic acid tolerant traits. As a result, three QTLs were located on chromosomes 9, 12, and 16, respectively, which explained 38.8-65.9 % of the range of phenotypic variation. Furthermore, twelve genes of the candidates fell into the three QTL regions by integrating the QTL analysis with candidates of acetic acid tolerant genes. These results provided a novel avenue to obtain more robust strains.

  9. Phosphoproteomic analysis of the Chlamydia caviae elementary body and reticulate body forms

    PubMed Central

    Adams, Nancy E.; Maurelli, Anthony T.

    2015-01-01

    Chlamydia are Gram-negative, obligate intracellular bacteria responsible for significant diseases in humans and economically important domestic animals. These pathogens undergo a unique biphasic developmental cycle transitioning between the environmentally stable elementary body (EB) and the replicative intracellular reticulate body (RB), a conversion that appears to require extensive regulation of protein synthesis and function. However, Chlamydia possess a limited number of canonical mechanisms of transcriptional regulation. Ser/Thr/Tyr phosphorylation of proteins in bacteria has been increasingly recognized as an important mechanism of post-translational control of protein function. We utilized 2D gel electrophoresis coupled with phosphoprotein staining and MALDI-TOF/TOF analysis to map the phosphoproteome of the EB and RB forms of Chlamydia caviae. Forty-two non-redundant phosphorylated proteins were identified (some proteins were present in multiple locations within the gels). Thirty-four phosphorylated proteins were identified in EBs, including proteins found in central metabolism and protein synthesis, Chlamydia-specific hypothetical proteins and virulence-related proteins. Eleven phosphorylated proteins were identified in RBs, mostly involved in protein synthesis and folding and a single virulence-related protein. Only three phosphoproteins were found in both EB and RB phosphoproteomes. Collectively, 41 of 42 C. caviae phosphoproteins were present across Chlamydia species, consistent with the existence of a conserved chlamydial phosphoproteome. The abundance of stage-specific phosphoproteins suggests that protein phosphorylation may play a role in regulating the function of developmental-stage-specific proteins and/or may function in concert with other factors in directing EB–RB transitions. PMID:25998263

  10. Phosphoproteomic analysis of the Chlamydia caviae elementary body and reticulate body forms.

    PubMed

    Fisher, Derek J; Adams, Nancy E; Maurelli, Anthony T

    2015-08-01

    Chlamydia are Gram-negative, obligate intracellular bacteria responsible for significant diseases in humans and economically important domestic animals. These pathogens undergo a unique biphasic developmental cycle transitioning between the environmentally stable elementary body (EB) and the replicative intracellular reticulate body (RB), a conversion that appears to require extensive regulation of protein synthesis and function. However, Chlamydia possess a limited number of canonical mechanisms of transcriptional regulation. Ser/Thr/Tyr phosphorylation of proteins in bacteria has been increasingly recognized as an important mechanism of post-translational control of protein function. We utilized 2D gel electrophoresis coupled with phosphoprotein staining and MALDI-TOF/TOF analysis to map the phosphoproteome of the EB and RB forms of Chlamydia caviae. Forty-two non-redundant phosphorylated proteins were identified (some proteins were present in multiple locations within the gels). Thirty-four phosphorylated proteins were identified in EBs, including proteins found in central metabolism and protein synthesis, Chlamydia-specific hypothetical proteins and virulence-related proteins. Eleven phosphorylated proteins were identified in RBs, mostly involved in protein synthesis and folding and a single virulence-related protein. Only three phosphoproteins were found in both EB and RB phosphoproteomes. Collectively, 41 of 42 C. caviae phosphoproteins were present across Chlamydia species, consistent with the existence of a conserved chlamydial phosphoproteome. The abundance of stage-specific phosphoproteins suggests that protein phosphorylation may play a role in regulating the function of developmental-stage-specific proteins and/or may function in concert with other factors in directing EB-RB transitions.

  11. Picking the right tool for the job--Phosphoproteomics of egg activation.

    PubMed

    Wessel, Gary M

    2015-12-01

    Eggs are the rarest cell in the human body, yet their study is essential for the fields of fertility, reproduction, and fetal health. Guo et al. (Proteomics 2015, 15, 4080-4095) use a "surrogate" animal to discover the phosphoproteomic pathways involved in egg activation. With datasets of several thousand phosphosites on 2500 different proteins, these investigators have defined new pathways, connections to pathways, and priorities in searches for how eggs are activated at fertilization. These results in a sea urchin are now transposable to mammals for testing on a per candidate strategy. PMID:26573262

  12. Picking the right tool for the job--Phosphoproteomics of egg activation.

    PubMed

    Wessel, Gary M

    2015-12-01

    Eggs are the rarest cell in the human body, yet their study is essential for the fields of fertility, reproduction, and fetal health. Guo et al. (Proteomics 2015, 15, 4080-4095) use a "surrogate" animal to discover the phosphoproteomic pathways involved in egg activation. With datasets of several thousand phosphosites on 2500 different proteins, these investigators have defined new pathways, connections to pathways, and priorities in searches for how eggs are activated at fertilization. These results in a sea urchin are now transposable to mammals for testing on a per candidate strategy.

  13. The diagnosis and management of aortic dissection.

    PubMed

    Karthikesalingam, A; Holt, P J E; Hinchliffe, R J; Thompson, M M; Loftus, I M

    2010-04-01

    Aortic dissection represents the most common aortic emergency, affecting 3 to 4 per 100,000 people per year and is still associated with a high mortality. Twenty percent of the patients with aortic dissection die before reaching hospital and 30% die during hospital admission. Aortic dissections may be classified in 3 ways: according to their anatomical extent (the Stanford or DeBakey systems), according to the time from onset (acute or chronic), and according to the underlying pathology (the European Society of Cardiologists' system). Advances in endovascular technology have provided new treatment options. Hybrid endovascular and conventional open surgical repair represent the mainstay of treatment for acute type A dissection. Medical management remains the gold standard for acute and uncomplicated chronic type B dissection, though endovascular surgery offers exciting potential in the management of complicated type B dissection through sealing of the intimal entry tear.

  14. [Isolated spontaneous dissection of visceral arteries].

    PubMed

    Corral, M A; Encinas, J; Fernández-Pérez, G C

    2014-01-01

    We present the cases of two men with isolated spontaneous dissection of visceral arteries diagnosed by multidetector CT. In the first patient, dissection of the celiac trunk was associated with periarterial changes. In the second patient, dissection of the superior mesenteric artery was associated with stenosis at the root of the celiac trunk. Both patients presented with acute pain, which was more intense and longer lasting in the first patient. Aortic dissection was suspected clinically in both patients. Both dissections were short and had patent saccular false lumens and reduced caliber of the true lumens. This morphological type is one of the most uncommon within this rare entity. However, in recent years, the number of cases published is rising. This suggests that this entity may have been underdiagnosed before the widespread use of multidetector CT. We discuss the two morphological classifications of dissection of the visceral arteries and the need to adapt therapeutic management to the particular circumstances of each case. PMID:21724211

  15. Intraoperative coronary artery dissection in fibromuscular dysplasia.

    PubMed

    Lou, Xiaoying; Mitter, Sumeet S; Blair, John E; Benzuly, Keith; Gambardella, Ivancarmine; Malaisrie, S Chris

    2015-04-01

    A 61-year-old woman with bicuspid aortic stenosis, an ascending aortic aneurysm, and a remote history of renal fibromuscular dysplasia underwent aortic root replacement complicated by extensive dissection of the left circumflex artery extending retrograde into the left anterior descending artery. This was managed by coronary artery bypass grafting, left ventricular support, and percutaneous coronary intervention for propagation of the dissection. This case highlights the prevalence, diagnosis, and management of intraoperative coronary dissection secondary to fibromuscular dysplasia. PMID:25841833

  16. SELPHI: correlation-based identification of kinase-associated networks from global phospho-proteomics data sets

    PubMed Central

    Petsalaki, Evangelia; Helbig, Andreas O.; Gopal, Anjali; Pasculescu, Adrian; Roth, Frederick P.; Pawson, Tony

    2015-01-01

    While phospho-proteomics studies have shed light on the dynamics of cellular signaling, they mainly describe global effects and rarely explore mechanistic details, such as kinase/substrate relationships. Tools and databases, such as NetworKIN and PhosphoSitePlus, provide valuable regulatory details on signaling networks but rely on prior knowledge. They therefore provide limited information on less studied kinases and fewer unexpected relationships given that better studied signaling events can mask condition- or cell-specific ‘network wiring’. SELPHI is a web-based tool providing in-depth analysis of phospho-proteomics data that is intuitive and accessible to non-bioinformatics experts. It uses correlation analysis of phospho-sites to extract kinase/phosphatase and phospho-peptide associations, and highlights the potential flow of signaling in the system under study. We illustrate SELPHI via analysis of phospho-proteomics data acquired in the presence of erlotinib—a tyrosine kinase inhibitor (TKI)—in cancer cells expressing TKI-resistant and -sensitive variants of the Epidermal Growth Factor Receptor. In this data set, SELPHI revealed information overlooked by the reporting study, including the known role of MET and EPHA2 kinases in conferring resistance to erlotinib in TKI sensitive strains. SELPHI can significantly enhance the analysis of phospho-proteomics data contributing to improved understanding of sample-specific signaling networks. SELPHI is freely available via http://llama.mshri.on.ca/SELPHI. PMID:25948583

  17. Evaluation of Educator & Student Use of & Attitudes toward Dissection & Dissection Alternatives

    ERIC Educational Resources Information Center

    Osenkowski, Pamela; Green, Che; Tjaden, Anne; Cunniff, Peggy

    2015-01-01

    Animal dissection has been routinely practiced in American biology classrooms for decades. With technological advancements, more states adopting student choice measures, and increased awareness about ethical concerns surrounding dissection, many useful dissection alternatives have been developed. To understand the current use of animal dissection…

  18. Identification of the PLK2-dependent phosphopeptidome by quantitative proteomics [corrected].

    PubMed

    Franchin, Cinzia; Cesaro, Luca; Pinna, Lorenzo A; Arrigoni, Giorgio; Salvi, Mauro

    2014-01-01

    Polo-like kinase 2 (PLK2) has been recently recognized as the major enzyme responsible for phosphorylation of α-synuclein at S129 in vitro and in vivo, suggesting that this kinase may play a key role in the pathogenesis of Parkinson's disease and other synucleinopathies. Moreover PLK2 seems to be implicated in cell division, oncogenesis, and synaptic regulation of the brain. However little is known about the phosphoproteome generated by PLK2 and, consequently the overall impact of PLK2 on cellular signaling. To fill this gap we exploited an approach based on in vitro kinase assay and quantitative phosphoproteomics. A proteome-derived peptide library obtained by digestion of undifferentiated human neuroblastoma cell line was exhaustively dephosphorylated by lambda phosphatase followed by incubation with or without PLK2 recombinant kinase. Stable isotope labeling based quantitative phosphoproteomics was applied to identify the phosphosites generated by PLK2. A total of 98 unique PLK2-dependent phosphosites from 89 proteins were identified by LC-MS/MS. Analysis of the primary structure of the identified phosphosites allowed the detailed definition of the kinase specificity and the compilation of a list of potential PLK2 targets among those retrieved in PhosphositePlus, a curated database of in cell/vivo phosphorylation sites.

  19. Large-Scale Phosphoproteomics Analysis of Whole Saliva Reveals a Distinct Phosphorylation Pattern

    PubMed Central

    Stone, Matthew D.; Chen, Xiaobing; McGowan, Thomas; Bandhakavi, Sricharan; Cheng, Bin; Rhodus, Nelson L.; Griffin, Timothy J.

    2011-01-01

    In-depth knowledge of bodily fluid phosphoproteomes, such as whole saliva, is limited. To better understand the whole saliva phosphoproteome, we generated a large-scale catalog of phosphorylated proteins. To circumvent the wide dynamic range of phosphoprotein abundance in whole saliva, we combined dynamic range compression using hexapeptide beads, strong cation exchange HPLC peptide fractionation, and immobilized metal affinity chromatography prior to mass spectrometry. In total, 217 unique phosphopeptides sites were identified representing 85 distinct phosphoproteins at 2.3% global FDR. From these peptides, 129 distinct phosphorylation sites were identified of which 57 were previously known, but only 11 of which had been previously identified in whole saliva. Cellular localization analysis revealed salivary phosphoproteins had a distribution similar to all known salivary proteins, but with less relative representation in “extracellular” and “plasma membrane” categories compared to salivary glycoproteins. Sequence alignment showed that phosphorylation occurred at acidic-directed kinase, proline-directed, and basophilic motifs. This differs from plasma phosphoproteins, which predominantly occur at Golgi casein kinase recognized sequences. Collectively, these results suggest diverse functions for salivary phosphoproteins and multiple kinases involved in their processing and secretion. In all, this study should lay groundwork for future elucidation of the functions of salivary protein phosphorylation. PMID:21299198

  20. NeuCode Labeling in Nematodes: Proteomic and Phosphoproteomic Impact of Ascaroside Treatment in Caenorhabditis elegans.

    PubMed

    Rhoads, Timothy W; Prasad, Aman; Kwiecien, Nicholas W; Merrill, Anna E; Zawack, Kelson; Westphall, Michael S; Schroeder, Frank C; Kimble, Judith; Coon, Joshua J

    2015-11-01

    The nematode Caenorhabditis elegans is an important model organism for biomedical research. We previously described NeuCode stable isotope labeling by amino acids in cell culture (SILAC), a method for accurate proteome quantification with potential for multiplexing beyond the limits of traditional stable isotope labeling by amino acids in cell culture. Here we apply NeuCode SILAC to profile the proteomic and phosphoproteomic response of C. elegans to two potent members of the ascaroside family of nematode pheromones. By consuming labeled E. coli as part of their diet, C. elegans nematodes quickly and easily incorporate the NeuCode heavy lysine isotopologues by the young adult stage. Using this approach, we report, at high confidence, one of the largest proteomic and phosphoproteomic data sets to date in C. elegans: 6596 proteins at a false discovery rate ≤ 1% and 6620 phosphorylation isoforms with localization probability ≥75%. Our data reveal a post-translational signature of pheromone sensing that includes many conserved proteins implicated in longevity and response to stress.

  1. Comprehensive Analysis of the Membrane Phosphoproteome Regulated by Oligogalacturonides in Arabidopsis thaliana

    PubMed Central

    Mattei, Benedetta; Spinelli, Francesco; Pontiggia, Daniela; De Lorenzo, Giulia

    2016-01-01

    Early changes in the Arabidopsis thaliana membrane phosphoproteome in response to oligogalacturonides (OGs), a class of plant damage-associated molecular patterns (DAMPs), were analyzed by two complementary proteomic approaches. Differentially phosphorylated sites were determined through phosphopeptide enrichment followed by LC-MS/MS using label-free quantification; differentially phosphorylated proteins were identified by 2D-DIGE combined with phospho-specific fluorescent staining (phospho-DIGE). This large-scale phosphoproteome analysis of early OG-signaling enabled us to determine 100 regulated phosphosites using LC-MS/MS and 46 differential spots corresponding to 34 pdhosphoproteins using phospho-DIGE. Functional classification showed that the OG-responsive phosphoproteins include kinases, phosphatases and receptor-like kinases, heat shock proteins (HSPs), reactive oxygen species (ROS) scavenging enzymes, proteins related to cellular trafficking, transport, defense and signaling as well as novel candidates for a role in immunity, for which elicitor-induced phosphorylation changes have not been shown before. A comparison with previously identified elicitor-regulated phosphosites shows only a very limited overlap, uncovering the immune-related regulation of 70 phosphorylation sites and revealing novel potential players in the regulation of elicitor-dependent immunity. PMID:27532006

  2. Large scale phosphoproteome analysis of LNCaP human prostate cancer cells.

    PubMed

    Myung, Jae-Kyung; Sadar, Marianne D

    2012-08-01

    Prostate cancer is the most frequently diagnosed cancer among men in the western world. The androgen receptor, a phosphoprotein, is suspected to be involved in all stages of the prostate cancer. Androgen receptor activity can be modulated by various kinases such as PKA, MAPK, AKT, and Src. Phosphorylation is an important post-translational modification and serves as a molecular on-off switch to regulate signaling. Disruptions of cellular phosphorylation are associated with various diseases such as cancer and kinases provide important drug targets. Here we present an analysis of the phosphoproteome in LNCaP human prostate cancer cells. The analytical strategy employed here used proteomics based methodologies with a combination of detergents and chaotropic reagents during trypsin digestion followed by titanium dioxide enrichment of phosphopeptides. Over the course of multiple analyses by mass spectrometry we identified a total of 746 phosphorylation sites in 540 phosphopeptides corresponding to 116 phosphoproteins, of which 56 had not been previously reported. Phosphoproteins identified included transcription factors, co-regulators of the androgen receptor, and cancer-related proteins that include β-catenin, USP10, and histone deacetylase-2. The information of signaling pathways, motifs of phosphorylated peptides, biological processes, molecular functions, cellular components, and protein interactions from the identified phosphoproteins established a map of phosphoproteome and signaling pathways in LNCaP cells.

  3. The global phosphoproteome of Chlamydomonas reinhardtii reveals complex organellar phosphorylation in the flagella and thylakoid membrane.

    PubMed

    Wang, Hongxia; Gau, Brian; Slade, William O; Juergens, Matthew; Li, Ping; Hicks, Leslie M

    2014-09-01

    Chlamydomonas reinhardtii is the most intensively-studied and well-developed model for investigation of a wide-range of microalgal processes ranging from basic development through understanding triacylglycerol production. Although proteomic technologies permit interrogation of these processes at the protein level and efforts to date indicate phosphorylation-based regulation of proteins in C. reinhardtii is essential for its underlying biology, characterization of the C. reinhardtii phosphoproteome has been limited. Herein, we report the richest exploration of the C. reinhardtii proteome to date. Complementary enrichment strategies were used to detect 4588 phosphoproteins distributed among every cellular component in C. reinhardtii. Additionally, we report 18,160 unique phosphopeptides at <1% false discovery rate, which comprise 15,862 unique phosphosites - 98% of which are novel. Given that an estimated 30% of proteins in a eukaryotic cell are subject to phosphorylation, we report the majority of the phosphoproteome (23%) of C. reinhardtii. Proteins in key biological pathways were phosphorylated, including photosynthesis, pigment production, carbon assimilation, glycolysis, and protein and carbohydrate metabolism, and it is noteworthy that hyperphosphorylation was observed in flagellar proteins. This rich data set is available via ProteomeXchange (ID: PXD000783) and will significantly enhance understanding of a range of regulatory mechanisms controlling a variety of cellular process and will serve as a critical resource for the microalgal community.

  4. Comprehensive Analysis of the Membrane Phosphoproteome Regulated by Oligogalacturonides in Arabidopsis thaliana.

    PubMed

    Mattei, Benedetta; Spinelli, Francesco; Pontiggia, Daniela; De Lorenzo, Giulia

    2016-01-01

    Early changes in the Arabidopsis thaliana membrane phosphoproteome in response to oligogalacturonides (OGs), a class of plant damage-associated molecular patterns (DAMPs), were analyzed by two complementary proteomic approaches. Differentially phosphorylated sites were determined through phosphopeptide enrichment followed by LC-MS/MS using label-free quantification; differentially phosphorylated proteins were identified by 2D-DIGE combined with phospho-specific fluorescent staining (phospho-DIGE). This large-scale phosphoproteome analysis of early OG-signaling enabled us to determine 100 regulated phosphosites using LC-MS/MS and 46 differential spots corresponding to 34 pdhosphoproteins using phospho-DIGE. Functional classification showed that the OG-responsive phosphoproteins include kinases, phosphatases and receptor-like kinases, heat shock proteins (HSPs), reactive oxygen species (ROS) scavenging enzymes, proteins related to cellular trafficking, transport, defense and signaling as well as novel candidates for a role in immunity, for which elicitor-induced phosphorylation changes have not been shown before. A comparison with previously identified elicitor-regulated phosphosites shows only a very limited overlap, uncovering the immune-related regulation of 70 phosphorylation sites and revealing novel potential players in the regulation of elicitor-dependent immunity. PMID:27532006

  5. Phosphoproteomic analysis of differentiating Leishmania parasites reveals a unique stage-specific phosphorylation motif.

    PubMed

    Tsigankov, Polina; Gherardini, Pier Federico; Helmer-Citterich, Manuela; Späth, Gerald F; Zilberstein, Dan

    2013-07-01

    Protists of the genus Leishmania are obligatory intracellular parasites that cause a wide range of cutaneous, mucocutaneous, and visceral diseases in humans. They cycle between phagolysosomes of mammalian macrophages and the sand fly midgut, proliferating as intracellular amastigotes and extracellular promastigotes, respectively. Exposure to a lysosomal environment, i.e. acidic pH and body temperature, signals promastigotes to differentiate into amastigotes. Time course analyses indicated that Leishmania differentiation is a highly regulated and coordinated process. However, the role of posttranslational events such as protein phosphorylation in this process is still unknown. Herein, we analyzed and compared the phosphoproteomes of L. donovani amastigotes and promastigotes using an axenic host-free system that simulates parasite differentiation. Shotgun phosphopeptide analysis revealed 1614 phosphorylation residues (p-sites) corresponding to 627 proteins. The analysis indicated that the majority of the p-sites are stage-specific. Serine phosphorylation in a previously identified trypanosomatid-specific "SF" motif was significantly enriched in amastigotes. We identified a few phosophotyrosines (pY), mostly in proteins known to participate in signal transduction pathways. The analysis indicated that Leishmania contains proteins with multiple p-sites that are phosphorylated at distinct stages of the life cycle. For over half of the phosphorylation events, changes in phosphoprotein abundance did not positively correlate with changes in protein abundance, suggesting functional regulation. This study compares, for the first time, the phosphoproteins of L. donovani axenic promastigotes and amastigotes and provides the largest data set of the Leishmania phosphoproteome to date.

  6. Comprehensive Analysis of the Membrane Phosphoproteome Regulated by Oligogalacturonides in Arabidopsis thaliana.

    PubMed

    Mattei, Benedetta; Spinelli, Francesco; Pontiggia, Daniela; De Lorenzo, Giulia

    2016-01-01

    Early changes in the Arabidopsis thaliana membrane phosphoproteome in response to oligogalacturonides (OGs), a class of plant damage-associated molecular patterns (DAMPs), were analyzed by two complementary proteomic approaches. Differentially phosphorylated sites were determined through phosphopeptide enrichment followed by LC-MS/MS using label-free quantification; differentially phosphorylated proteins were identified by 2D-DIGE combined with phospho-specific fluorescent staining (phospho-DIGE). This large-scale phosphoproteome analysis of early OG-signaling enabled us to determine 100 regulated phosphosites using LC-MS/MS and 46 differential spots corresponding to 34 pdhosphoproteins using phospho-DIGE. Functional classification showed that the OG-responsive phosphoproteins include kinases, phosphatases and receptor-like kinases, heat shock proteins (HSPs), reactive oxygen species (ROS) scavenging enzymes, proteins related to cellular trafficking, transport, defense and signaling as well as novel candidates for a role in immunity, for which elicitor-induced phosphorylation changes have not been shown before. A comparison with previously identified elicitor-regulated phosphosites shows only a very limited overlap, uncovering the immune-related regulation of 70 phosphorylation sites and revealing novel potential players in the regulation of elicitor-dependent immunity.

  7. Keeping Dissection Alive for Medical Students

    ERIC Educational Resources Information Center

    Chambers, James; Emlyn-Jones, Daniel

    2009-01-01

    Traditional dissection teaching is being reduced in a number of medical schools, particularly in the United Kingdom. In response to this, 12 medical students from Warwick University, UK, traveled to the Island of Grenada for an intensive extracurricular dissection course at St. George's University. This course not only benefited the host…

  8. Coronary artery dissection after blunt chest trauma

    PubMed Central

    Shamsi, Fahad; Tai, Javed Majid; Bokhari, Saira

    2014-01-01

    Blunt thoracic trauma may result in cardiac injuries ranging from simple arrhythmias to fatal cardiac rupture. Coronary artery dissection culminating in acute myocardial infarction (AMI) is rare after blunt chest trauma. Here we report a case of a 37-year-old man who had an AMI secondary to coronary dissection resulting from blunt chest trauma after involvement in a physical fight. PMID:25246456

  9. Quick Dissection of the Segmental Bronchi

    ERIC Educational Resources Information Center

    Nakajima, Yuji

    2010-01-01

    Knowledge of the three-dimensional anatomy of the bronchopulmonary segments is essential for respiratory medicine. This report describes a quick guide for dissecting the segmental bronchi in formaldehyde-fixed human material. All segmental bronchi are easy to dissect, and thus, this exercise will help medical students to better understand the…

  10. Objecting To Dissection: A College Student's Handbook.

    ERIC Educational Resources Information Center

    National Anti-Vivisection Society, Chicago, IL.

    In a number of states, students from kindergarten through high school have won the right to refuse to dissect or kill animals and the right to substitute an alternative project. This booklet was designed to help college science students take an ethical stand by refusing to participate in dissection exercises. The booklet begins with an overview of…

  11. Beyond Dissection: Innovative Tools for Biology Education.

    ERIC Educational Resources Information Center

    Larson, Sandra, Ed.

    This catalog lists resources available for classroom use in teaching about anatomy and physiology which are alternatives to dissection. The entries are provided under three main categories: (1) Whole Animal Dissection/Vivisection; (2) Animal Organ or System Anatomy and Physiology; and (3) Other, including animal behavior, biotechnology,…

  12. Spontaneous Coronary Artery Dissection with Cardiac Tamponade.

    PubMed

    Goh, Anne C H; Lundstrom, Robert J

    2015-10-01

    Spontaneous coronary artery dissection is a rare cause of acute coronary syndrome. Clinical presentation ranges from chest pain alone to ST-segment-elevation myocardial infarction, ventricular fibrillation, and sudden death. The treatment of patients with spontaneous coronary artery dissection is challenging because the disease pathophysiology is unclear, optimal treatment is unknown, and short- and long-term prognostic data are minimal. We report the case of a 70-year-old woman who presented with an acute ST-segment-elevation myocardial infarction secondary to a spontaneous dissection of the left anterior descending coronary artery. She was treated conservatively. Cardiac tamponade developed 16 hours after presentation. Repeat coronary angiography revealed extension of the dissection. Medical therapy was continued after the hemopericardium was aspirated. The patient remained asymptomatic 3 years after hospital discharge. To our knowledge, this is the first reported case of spontaneous coronary artery dissection in association with cardiac tamponade that was treated conservatively and had a successful outcome. PMID:26504447

  13. Novel aspects of grapevine response to phytoplasma infection investigated by a proteomic and phospho-proteomic approach with data integration into functional networks

    PubMed Central

    2013-01-01

    Background Translational and post-translational protein modifications play a key role in the response of plants to pathogen infection. Among the latter, phosphorylation is critical in modulating protein structure, localization and interaction with other partners. In this work, we used a multiplex staining approach with 2D gels to study quantitative changes in the proteome and phosphoproteome of Flavescence dorée-affected and recovered ‘Barbera’ grapevines, compared to healthy plants. Results We identified 48 proteins that differentially changed in abundance, phosphorylation, or both in response to Flavescence dorée phytoplasma infection. Most of them did not show any significant difference in recovered plants, which, by contrast, were characterized by changes in abundance, phosphorylation, or both for 17 proteins not detected in infected plants. Some enzymes involved in the antioxidant response that were up-regulated in infected plants, such as isocitrate dehydrogenase and glutathione S-transferase, returned to healthy-state levels in recovered plants. Others belonging to the same functional category were even down-regulated in recovered plants (oxidoreductase GLYR1 and ascorbate peroxidase). Our proteomic approach thus agreed with previously published biochemical and RT-qPCR data which reported down-regulation of scavenging enzymes and accumulation of H2O2 in recovered plants, possibly suggesting a role for this molecule in remission from infection. Fifteen differentially phosphorylated proteins (| ratio | > 2, p < 0.05) were identified in infected compared to healthy plants, including proteins involved in photosynthesis, response to stress and the antioxidant system. Many were not differentially phosphorylated in recovered compared to healthy plants, pointing to their specific role in responding to infection, followed by a return to a steady-state phosphorylation level after remission of symptoms. Gene ontology (GO) enrichment and statistical

  14. Rapid and reproducible single-stage phosphopeptide enrichment of complex peptide mixtures: application to general and phosphotyrosine-specific phosphoproteomics experiments.

    PubMed

    Kettenbach, Arminja N; Gerber, Scott A

    2011-10-15

    Reversible protein phosphorylation is an essential regulatory component of virtually every cellular process and is frequently dysregulated in cancer. However, significant analytical barriers persist that hamper the routine application of phosphoproteomics in translational settings. Here, we present a straightforward and reproducible approach for the broadscale analysis of protein phosphorylation that relies on a single phosphopeptide enrichment step using titanium dioxide microspheres from whole cell lysate digests and compared it to the well-established SCX-TiO(2) workflow for phosphopeptide purification on a proteome-wide scale. We demonstrate the scaleabilty of our approach from 200 μg to 5 mg of total NCI-H23 non-small cell lung adenocarcinoma cell lysate digest and determine its quantitative reproducibility by label-free analysis of phosphopeptide peak areas from replicate purifications (median CV: 20% RSD). Finally, we combine this approach with immunoaffinity phosphotyrosine enrichment, enabling the identification of 3168 unique nonredundant phosphotyrosine peptides in two LC-MS/MS runs from 8 mg of HeLa peptides, each with 80% phosphotyrosine selectivity, at a peptide FDR of 0.2%. Taken together, we establish and validate a robust approach for proteome-wide phosphorylation analysis in a variety of scenarios that is easy to implement in biomedical research and translational settings. PMID:21899308

  15. Videoendoscopic single-port axillary dissection

    PubMed Central

    Uras, Cihan; Aytac, Erman; Aydogan, Fatih

    2011-01-01

    Videoendoscopy is newly used in breast and axillary surgery. Single-port surgery is one of the newest methods of minimally invasive surgery. This report describes the first case of videoendoscopic single-port axillary dissection. In histopathological evaluation, 24 lymph nodes were identified and one node was infiltrated by the cancer cells. Videoendoscopic single-port axillary dissection is a precise and improvable technique. Single-port videoendoscopic axillary dissection could be more feasible with individual tools that will be designed for minimally invasive breast surgery. PMID:22022116

  16. Recent findings and technological advances in phosphoproteomics for cells and tissues.

    PubMed

    von Stechow, Louise; Francavilla, Chiara; Olsen, Jesper V

    2015-01-01

    Site-specific phosphorylation is a fast and reversible covalent post-translational modification that is tightly regulated in cells. The cellular machinery of enzymes that write, erase and read these modifications (kinases, phosphatases and phospho-binding proteins) is frequently deregulated in different diseases, including cancer. Large-scale studies of phosphoproteins - termed phosphoproteomics - strongly rely on the use of high-performance mass spectrometric instrumentation. This powerful technology has been applied to study a great number of phosphorylation-based phenotypes. Nevertheless, many technical and biological challenges have to be overcome to identify biologically relevant phosphorylation sites in cells and tissues. This review describes different technological strategies to identify and quantify phosphorylation sites with high accuracy, without significant loss of analysis speed and reproducibility in tissues and cells. Moreover, computational tools for analysis, integration and biological interpretation of phosphorylation events are discussed.

  17. Characterization of the human plasma phosphoproteome using linear ion trap mass spectrometry and multiple search engines.

    PubMed

    Carrascal, Montserrat; Gay, Marina; Ovelleiro, David; Casas, Vanessa; Gelpí, Emilio; Abian, Joaquin

    2010-02-01

    Major plasma protein families play different roles in blood physiology and hemostasis and in immunodefense. Other proteins in plasma can be involved in signaling as chemical messengers or constitute biological markers of the status of distant tissues. In this respect, the plasma phosphoproteome holds potentially relevant information on the mechanisms modulating these processes through the regulation of protein activity. In this work we describe for the first time a collection of phosphopeptides identified in human plasma using immunoaffinity separation of the seven major serum protein families from other plasma proteins, SCX fractionation, and TiO(2) purification prior to LC-MS/MS analysis. One-hundred and twenty-seven phosphosites in 138 phosphopeptides mapping 70 phosphoproteins were identified with FDR < 1%. A high-confidence collection of phosphosites was obtained using a combined search with the OMSSA, SEQUEST, and Phenyx search engines.

  18. Recent findings and technological advances in phosphoproteomics for cells and tissues

    PubMed Central

    von Stechow, Louise; Francavilla, Chiara; Olsen, Jesper V

    2015-01-01

    Site-specific phosphorylation is a fast and reversible covalent post-translational modification that is tightly regulated in cells. The cellular machinery of enzymes that write, erase and read these modifications (kinases, phosphatases and phospho-binding proteins) is frequently deregulated in different diseases, including cancer. Large-scale studies of phosphoproteins – termed phosphoproteomics – strongly rely on the use of high-performance mass spectrometric instrumentation. This powerful technology has been applied to study a great number of phosphorylation-based phenotypes. Nevertheless, many technical and biological challenges have to be overcome to identify biologically relevant phosphorylation sites in cells and tissues. This review describes different technological strategies to identify and quantify phosphorylation sites with high accuracy, without significant loss of analysis speed and reproducibility in tissues and cells. Moreover, computational tools for analysis, integration and biological interpretation of phosphorylation events are discussed. PMID:26400465

  19. Systematic profiling of the bacterial phosphoproteome reveals bacterium-specific features of phosphorylation.

    PubMed

    Lin, Miao-Hsia; Sugiyama, Naoyuki; Ishihama, Yasushi

    2015-09-15

    Protein phosphorylation is a crucial posttranslational modification for regulating cellular processes in bacteria; however, it has not been extensively studied because of technical difficulties in the enrichment of phosphopeptides. We devised an enrichment protocol that enabled the identification of >1000 phosphopeptides from a single bacterial sample. We discovered three high-confidence serine and threonine phosphorylation motifs, as well as 29 other motifs at various levels of confidence, from three distinct bacterial phosphoproteomes. We found that the proline-directed and basophilic phosphorylation motifs that are commonly enriched in eukaryotes were not observed in bacteria. Unlike eukaryotes, bacteria had a low occurrence of both phosphorylation and acetylation in N-terminal phosphopeptides. Because infection of host cells by bacterial pathogens is often accompanied by kinase-mediated phosphorylation events, the differences in phosphorylation preferences between bacteria and eukaryotes revealed by this study could be useful in identifying bacterial-specific targets for future therapies. PMID:26373674

  20. Unbiased phosphoproteomic method identifies the initial effects of a methacrylic acid copolymer on macrophages.

    PubMed

    Chamberlain, Michael Dean; Wells, Laura A; Lisovsky, Alexandra; Guo, Hongbo; Isserlin, Ruth; Talior-Volodarsky, Ilana; Mahou, Redouan; Emili, Andrew; Sefton, Michael V

    2015-08-25

    An unbiased phosphoproteomic method was used to identify biomaterial-associated changes in the phosphorylation patterns of macrophage-like cells. The phosphorylation differences between differentiated THP1 (dTHP1) cells treated for 10, 20, or 30 min with a vascular regenerative methacrylic acid (MAA) copolymer or a control methyl methacrylate (MM) copolymer were determined by MS. There were 1,470 peptides (corresponding to 729 proteins) that were differentially phosphorylated in dTHP1 cells treated with the two materials with a greater cellular response to MAA treatment. In addition to identifying pathways (such as integrin signaling and cytoskeletal arrangement) that are well known to change with cell-material interaction, previously unidentified pathways, such as apoptosis and mRNA splicing, were also discovered. PMID:26261332

  1. Automated Immobilized Metal Affinity Chromatography System for Enrichment of Escherichia coli Phosphoproteome

    SciTech Connect

    Qu, Yi; Wu, Si; Zhao, Rui; Zink, Erika M.; Orton, Daniel J.; Moore, Ronald J.; Meng, Da; Clauss, Therese RW; Aldrich, Joshua T.; Lipton, Mary S.; Pasa-Tolic, Ljiljana

    2013-06-05

    Enrichment of bacterial phosphopeptides is an essential step prior to bottom-up mass spectrometry-based analysis of the phosphoproteome, which is fundamental to understanding the role of phosphoproteins in cell signaling and regulation of protein activity. We developed an automated IMAC system to enrich strong cation exchange-fractionated phosphopeptides from the soluble proteome of Escherichia coli MG1655 grown on minimal medium. Initial demonstration of the system resulted in identification of 75 phosphopeptides covering 52 phosphoproteins. Consistent with previous studies, many of these phosphoproteins are involved in the carbohydrate portion of central metabolism. The automated system utilizes a large capacity IMAC column that can effectively enrich phosphopeptides from a bacterial sample by increasing peptide loading and reducing the wash time. An additional benefit of the automated IMAC system is reduced labor and associated costs.

  2. Systematic profiling of the bacterial phosphoproteome reveals bacterium-specific features of phosphorylation.

    PubMed

    Lin, Miao-Hsia; Sugiyama, Naoyuki; Ishihama, Yasushi

    2015-09-15

    Protein phosphorylation is a crucial posttranslational modification for regulating cellular processes in bacteria; however, it has not been extensively studied because of technical difficulties in the enrichment of phosphopeptides. We devised an enrichment protocol that enabled the identification of >1000 phosphopeptides from a single bacterial sample. We discovered three high-confidence serine and threonine phosphorylation motifs, as well as 29 other motifs at various levels of confidence, from three distinct bacterial phosphoproteomes. We found that the proline-directed and basophilic phosphorylation motifs that are commonly enriched in eukaryotes were not observed in bacteria. Unlike eukaryotes, bacteria had a low occurrence of both phosphorylation and acetylation in N-terminal phosphopeptides. Because infection of host cells by bacterial pathogens is often accompanied by kinase-mediated phosphorylation events, the differences in phosphorylation preferences between bacteria and eukaryotes revealed by this study could be useful in identifying bacterial-specific targets for future therapies.

  3. The beginnings of crop phosphoproteomics: exploring early warning systems of stress

    PubMed Central

    Rampitsch, Christof; Bykova, Natalia V.

    2012-01-01

    This review examines why a knowledge of plant protein phosphorylation events is important in devising strategies to protect crops from both biotic and abiotic stresses, and why proteomics should be included when studying stress pathways. Most of the achievements in elucidating phospho-signaling pathways in biotic and abiotic stress are reported from model systems: while these are discussed, this review attempts mainly to focus on work done with crops, with examples of achievements reported from rice, maize, wheat, grape, Brassica, tomato, and soy bean after cold acclimation, hormonal and oxidative hydrogen peroxide treatment, salt stress, mechanical wounding, or pathogen challenge. The challenges that remain to transfer this information into a format that can be used to protect crops against biotic and abiotic stresses are enormous. The tremendous increase in the speed and ease of DNA sequencing is poised to reveal the whole genomes of many crop species in the near future, which will facilitate phosphoproteomics and phosphogenomics research. PMID:22783265

  4. Plasma phosphoproteome and differential plasma phosphoproteins with opisthorchis viverrini-related cholangiocarcinoma.

    PubMed

    Kotawong, Kanawut; Thitapakorn, Veerachai; Roytrakul, Sittiruk; Phaonakrop, Narumon; Viyanant, Vithoon; Na-Bangchang, Kesara

    2015-01-01

    This study was conducted to investigate the plasma phosphoproteome and differential plasma phosphoproteins in cases of of Opisthorchis viverrini (OV)-related cholangiocarcinoma (CCA). Plasma phosphoproteomes from CCA patients (10) and non-CCA subjects (5 each for healthy subjects and OV infection) were investigated using gel-based and solution-based LC-MS/MS. Phosphoproteins in plasma samples were enriched and analyzed by LC-MS/MS. STRAP, PANTHER, iPath, and MeV programs were applied for the identification of their functions, signaling and metabolic pathways; and for the discrimination of potential biomarkers in CCA patients and non-CCA subjects, respectively. A total of 90 and 60 plasma phosphoproteins were identified by gel-based and solution-based LC-MS/MS, respectively. Most of the phosphoproteins were cytosol proteins which play roles in several cellular processes, signaling pathways, and metabolic pathways (STRAP, PANTHER, and iPath analysis). The absence of serine/arginine repetitive matrix protein 3 (A6NNA2), tubulin tyrosine ligase-like family, member 6, and biorientation of chromosomes in cell division protein 1-like (Q8NFC6) in plasma phosphoprotein were identified as potential biomarkers for the differentiation of healthy subjects from patients with CCA and OV infection. To differentiate CCA from OV infection, the absence of both serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit beta isoform and coiled-coil domain-containing protein 126 precursor (Q96EE4) were then applied. A combination of 5 phosphoproteins may new alternative choices for CCA diagnosis. PMID:25735322

  5. Phosphoproteomic Profiling of Human Myocardial Tissues Distinguishes Ischemic from Non-Ischemic End Stage Heart Failure

    PubMed Central

    Njoroge, Linda W.; Thompson, J. Will; Soderblom, Erik J.; Feger, Bryan J.; Troupes, Constantine D.; Hershberger, Kathleen A.; Ilkayeva, Olga R.; Nagel, Whitney L.; Landinez, Gina P.; Shah, Kishan M.; Burns, Virginia A.; Santacruz, Lucia; Hirschey, Matthew D.; Foster, Matthew W.; Milano, Carmelo A.; Moseley, M. Arthur; Piacentino, Valentino; Bowles, Dawn E.

    2014-01-01

    The molecular differences between ischemic (IF) and non-ischemic (NIF) heart failure are poorly defined. A better understanding of the molecular differences between these two heart failure etiologies may lead to the development of more effective heart failure therapeutics. In this study extensive proteomic and phosphoproteomic profiles of myocardial tissue from patients diagnosed with IF or NIF were assembled and compared. Proteins extracted from left ventricular sections were proteolyzed and phosphopeptides were enriched using titanium dioxide resin. Gel- and label-free nanoscale capillary liquid chromatography coupled to high resolution accuracy mass tandem mass spectrometry allowed for the quantification of 4,436 peptides (corresponding to 450 proteins) and 823 phosphopeptides (corresponding to 400 proteins) from the unenriched and phospho-enriched fractions, respectively. Protein abundance did not distinguish NIF from IF. In contrast, 37 peptides (corresponding to 26 proteins) exhibited a ≥2-fold alteration in phosphorylation state (p<0.05) when comparing IF and NIF. The degree of protein phosphorylation at these 37 sites was specifically dependent upon the heart failure etiology examined. Proteins exhibiting phosphorylation alterations were grouped into functional categories: transcriptional activation/RNA processing; cytoskeleton structure/function; molecular chaperones; cell adhesion/signaling; apoptosis; and energetic/metabolism. Phosphoproteomic analysis demonstrated profound post-translational differences in proteins that are involved in multiple cellular processes between different heart failure phenotypes. Understanding the roles these phosphorylation alterations play in the development of NIF and IF has the potential to generate etiology-specific heart failure therapeutics, which could be more effective than current therapeutics in addressing the growing concern of heart failure. PMID:25117565

  6. A rare cause of recurrent aortic dissection.

    PubMed

    Agrawal, Yashwant; Gupta, Vishal

    2016-07-01

    We report the case of a 19-year-old man with a history of Loeys-Dietz syndrome (LDS), which was diagnosed when he had a Stanford type A aortic dissection. He also had multiple aneurysms including ones in the innominate, right common carotid, and right internal mammary arteries. He had had multiple procedures including Bentall's procedure, repeat sternotomy with complete arch and valve replacement, and coil embolization of internal mammary artery aneurysm in the past. His LDS was characterized by gene mutation for transforming growth factor-β receptor 1. He presented to our facility with sudden onset of back pain, radiating to the right shoulder and chest. He was diagnosed with Stanford type B aortic dissection and underwent thoracic aorta endovascular repair for his aortic dissection. This case represents the broad spectrum of pathology associated with LDS where even with regular surveillance and aggressive medical management the patient developed Stanford B aortic dissection. PMID:27358537

  7. A New Idea for Dissecting Tray

    ERIC Educational Resources Information Center

    Branham, Arthur

    1976-01-01

    A method of preparing a special dissecting tray to be used with transmitted light as well as reflected light is presented. It may also be used with an overhead projector to illustrate some skeletal structures in vertebrates. (Author/EB)

  8. Animal Rights Groups Target High School Dissection.

    ERIC Educational Resources Information Center

    Trotter, Andrew

    1992-01-01

    Two groups leading the charge against dissection are People for the Ethical Treatment of Animals (PETA) and the Student Action Corps for Animals (SACA). Protests by student and community members remain the movement's strongest weapon. (MLF)

  9. Structural insight into effector proteins of Gram-negative bacterial pathogens that modulate the phosphoproteome of their host

    PubMed Central

    Grishin, Andrey M; Beyrakhova, Ksenia A; Cygler, Miroslaw

    2015-01-01

    Invading pathogens manipulate cellular process of the host cell to establish a safe replicative niche. To this end they secrete a spectrum of proteins called effectors that modify cellular environment through a variety of mechanisms. One of the most important mechanisms is the manipulation of cellular signaling through modifications of the cellular phosphoproteome. Phosphorylation/dephosphorylation plays a pivotal role in eukaryotic cell signaling, with ∼500 different kinases and ∼130 phosphatases in the human genome. Pathogens affect the phosphoproteome either directly through the action of bacterial effectors, and/or indirectly through downstream effects of host proteins modified by the effectors. Here we review the current knowledge of the structure, catalytic mechanism and function of bacterial effectors that modify directly the phosphorylation state of host proteins. These effectors belong to four enzyme classes: kinases, phosphatases, phospholyases and serine/threonine acetylases. PMID:25565677

  10. The phosphoproteome in regenerating protoplasts from Physcomitrella patens protonemata shows changes paralleling postembryonic development in higher plants.

    PubMed

    Wang, Xiaoqin; Qi, Meiyan; Li, Jingyun; Ji, Zhongzhong; Hu, Yong; Bao, Fang; Mahalingam, Ramamurthy; He, Yikun

    2014-05-01

    The moss Physcomitrella patens is an ideal model plant to study plant developmental processes. To better understand the mechanism of protoplast regeneration, a phosphoproteome analysis was performed. Protoplasts were prepared from protonemata. By 4 d of protoplast regeneration, the first cell divisions had ensued. Through a highly selective titanium dioxide (TiO2)-based phosphopeptide enrichment method and mass spectrometric technology, more than 300 phosphoproteins were identified as protoplast regeneration responsive. Of these, 108 phosphoproteins were present on day 4 but not in fresh protoplasts or those cultured for 2 d. These proteins are catalogued here. They were involved in cell-wall metabolism, transcription, signal transduction, cell growth/division, and cell structure. These protein functions are related to cell morphogenesis, organogenesis, and development adjustment. This study presents a comprehensive analysis of phosphoproteome involved in protoplast regeneration and indicates that the mechanism of plant protoplast regeneration is similar to that of postembryonic development.

  11. Peripartum presentation of an acute aortic dissection.

    PubMed

    Lewis, S; Ryder, I; Lovell, A T

    2005-04-01

    We report the case of an acute type A aortic dissection occurring in a 35-year-old parturient. The initial diagnosis was missed; a subsequent emergency Caesarean section 3 weeks after presentation was followed by the development of left ventricular failure and pulmonary oedema in the early postoperative period. Echocardiography confirmed the diagnosis of aortic dissection and the patient underwent a successful surgical repair.

  12. Pulmonary hypertension and pulmonary artery dissection

    PubMed Central

    Corrêa, Ricardo de Amorim; Silva, Luciana Cristina dos Santos; Rezende, Cláudia Juliana; Bernardes, Rodrigo Castro; Prata, Tarciane Aline; Silva, Henrique Lima

    2013-01-01

    Pulmonary artery dissection is a fatal complication of long-standing pulmonary hypertension, manifesting as acute, stabbing chest pain, progressive dyspnea, cardiogenic shock, or sudden death. Its incidence has been underestimated, and therapeutic options are still scarce. In patients with pulmonary hypertension, new chest pain, acute chest pain, or cardiogenic shock should raise the suspicion of pulmonary artery dissection, which can result in sudden death. PMID:23670510

  13. Peripartum presentation of an acute aortic dissection.

    PubMed

    Lewis, S; Ryder, I; Lovell, A T

    2005-04-01

    We report the case of an acute type A aortic dissection occurring in a 35-year-old parturient. The initial diagnosis was missed; a subsequent emergency Caesarean section 3 weeks after presentation was followed by the development of left ventricular failure and pulmonary oedema in the early postoperative period. Echocardiography confirmed the diagnosis of aortic dissection and the patient underwent a successful surgical repair. PMID:15640303

  14. Searching for Novel Cdk5 Substrates in Brain by Comparative Phosphoproteomics of Wild Type and Cdk5−/− Mice

    PubMed Central

    Contreras-Vallejos, Erick; Utreras, Elías; Bórquez, Daniel A.; Prochazkova, Michaela; Terse, Anita; Jaffe, Howard; Toledo, Andrea; Arruti, Cristina; Pant, Harish C.; Kulkarni, Ashok B.; González-Billault, Christian

    2014-01-01

    Protein phosphorylation is the most common post-translational modification that regulates several pivotal functions in cells. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase which is mostly active in the nervous system. It regulates several biological processes such as neuronal migration, cytoskeletal dynamics, axonal guidance and synaptic plasticity among others. In search for novel substrates of Cdk5 in the brain we performed quantitative phosphoproteomics analysis, isolating phosphoproteins from whole brain derived from E18.5 Cdk5+/+ and Cdk5−/− embryos, using an Immobilized Metal-Ion Affinity Chromatography (IMAC), which specifically binds to phosphorylated proteins. The isolated phosphoproteins were eluted and isotopically labeled for relative and absolute quantitation (iTRAQ) and mass spectrometry identification. We found 40 proteins that showed decreased phosphorylation at Cdk5−/− brains. In addition, out of these 40 hypophosphorylated proteins we characterized two proteins, :MARCKS (Myristoylated Alanine-Rich protein Kinase C substrate) and Grin1 (G protein regulated inducer of neurite outgrowth 1). MARCKS is known to be phosphorylated by Cdk5 in chick neural cells while Grin1 has not been reported to be phosphorylated by Cdk5. When these proteins were overexpressed in N2A neuroblastoma cell line along with p35, serine phosphorylation in their Cdk5 motifs was found to be increased. In contrast, treatments with roscovitine, the Cdk5 inhibitor, resulted in an opposite effect on serine phosphorylation in N2A cells and primary hippocampal neurons transfected with MARCKS. In summary, the results presented here identify Grin 1 as novel Cdk5 substrate and confirm previously identified MARCKS as a a bona fide Cdk5 substrate. PMID:24658276

  15. The Effect of Animal Dissections on Student Acquisition of Knowledge of and Attitudes toward the Animals Dissected.

    ERIC Educational Resources Information Center

    McCollum, Terry L.

    A conflict exists over the use of animals in the classroom. One aspect of this use involved the dissection of animals. Animal protection advocates report that dissections constitute abuse of the animals dissected. The advocates state that what is learned by dissection could be more effectively learned by other means. Some science educators state…

  16. Molecular Dissection of Phage Endolysin

    PubMed Central

    Pohane, Amol Arunrao; Joshi, Himanshu; Jain, Vikas

    2014-01-01

    Mycobacterium tuberculosis has always been recognized as one of the most successful pathogens. Bacteriophages that attack and kill mycobacteria offer an alternate mechanism for the curtailment of this bacterium. Upon infection, mycobacteriophages produce lysins that catalyze cell wall peptidoglycan hydrolysis and mycolic acid layer breakdown of the host resulting in bacterial cell rupture and virus release. The ability to lyse bacterial cells make lysins extremely significant. We report here a detailed molecular dissection of the function and regulation of mycobacteriophage D29 Lysin A. Several truncated versions of Lysin A were constructed, and their activities were analyzed by zymography and by expressing them in both Escherichia coli and Mycobacterium smegmatis. Our experiments establish that Lysin A harbors two catalytically active domains, both of which show E. coli cell lysis upon their expression exclusively in the periplasmic space. However, the expression of only one of these domains and the full-length Lysin A caused M. smegmatis cell lysis. Interestingly, full-length protein remained inactive in E. coli periplasm. Our data suggest that the inactivity is ensued by a C-terminal domain that interacts with the N-terminal domain. This interaction was affirmed by surface plasmon resonance. Our experiments also demonstrate that the C-terminal domain of Lysin A selectively binds to M. tuberculosis and M. smegmatis peptidoglycans. Our methodology of studying E. coli cell lysis by Lysin A and its truncations after expressing these proteins in the bacterial periplasm with the help of signal peptide paves the way for a large scale identification and analysis of such proteins obtained from other bacteriophages. PMID:24627486

  17. Incomplete nested dissection for solving n by n grid problems

    NASA Technical Reports Server (NTRS)

    George, A.; Poole, W. G., Jr.; Voigt, R. G.

    1978-01-01

    Nested dissection orderings are known to be very effective for solving sparse positive definite linear systems which arise from n by n grid problems. In this paper we consider incomplete nested dissection, an ordering which corresponds to the premature termination of nested dissection. Analyses of the arithmetic and storage requirements for incomplete nested dissection are given and the ordering is shown to be competitive with nested dissection with regard to arithmetic operations and superior to that ordering in storage requirements.

  18. Dissection and dissection-associated required experiences improve student performance in gross anatomy: Differences among quartiles.

    PubMed

    Pizzimenti, Marc A; Pantazis, Nicholas; Sandra, Alexander; Hoffmann, Darren S; Lenoch, Susan; Ferguson, Kristi J

    2016-05-01

    To promote student learning, educational strategies should provide multiple levels of engagement with the subject matter. This study investigated examination data from five first year medical gross anatomy class cohorts (692 students) to determine if enhanced student performance was correlated with learning through dissection in a course that used a rotating dissection schedule coupled with peer teaching and other associated experiences. When students performed two of five weekly dissections for a given unit, their average scores on both laboratory and written examinations tended to increase as compared to when they had completed only one week of dissection (P < 0.01). However, these performance gains differed across the class strata and were related to the amount of dissection completed. Students in the upper quartile (UQS) of the class benefited when they had dissected once (92.8%) or twice (92.4%), and these scores were significantly higher than those attained when learning from peers (90.3%, P < 0.01). Students in the lower quartile (LQS) benefited most from the dissection experiences, where practical examination performance was better (77.8% and 80.5%) than when these students learned material from their peers (73.7%, P < 0.01). Although UQS benefited from dissection, LQS benefited to a greater extent in both the practical and written examinations with dissection. Although limited, these data suggest that dissection, coupled with associated educational activities, is an effective pedagogical strategy for learning. Further investigation is required to evaluate the concomitant benefits of peer teaching that are associated with the dissection experience. Anat Sci Educ 9: 238-246. © 2015 American Association of Anatomists.

  19. Student injuries in the dissecting room.

    PubMed

    Cornwall, Jon; Davies, Tilman M; Lees, David

    2013-01-01

    Cadaver dissection is the first opportunity for many students to practice handling human tissue and is their first exposure to the occupational hazards involved with this task. Few studies examine dissection room injuries to ascertain the dangers associated with dissecting. We performed a retrospective cohort analysis of dissection room injuries from four student cohorts over an eleven-year period (2001-2011), including second-year medical students, third-year medical students, second-year dental students, and third-year science students. Injury data included activity causing injury, object responsible, and injury site. A total of 163 injuries during 70,039 hours of dissection were recorded, with 66 in third-year medical students, 42 in second-year medical students, 36 in third-year science students, and 16 in second-year dental students. The overall rate was 2.87 injuries per 1,000 dissection hours, with second-year medical students most frequently injured (5.5 injuries per 1,000 hours); third-year medical students were least frequently injured (1.3 injuries per 1,000 hours). A significant difference in injury rates between student groups indicated a higher than expected injury rate to second-year medical students and lower than expected rates to third-year medical students. Injury rates increased for most groups between 2001-2006 and 2007-2011 periods. Most injuries (79%) were from scalpel cuts to the finger or thumb. This study provides injury rates for dissection room injuries to students, indicating differences in injury frequency between cohorts and an increase in injury rate over time. As scalpel cuts were the most likely injury mechanism, targeting scalpel handling with preventative strategies may reduce future injury risk.

  20. Science Teachers and the Dissection Debate: Perspectives on Animal Dissection and Alternatives

    ERIC Educational Resources Information Center

    Oakley, Jan

    2012-01-01

    This study investigated Ontario science and biology teachers' practices and attitudes toward animal dissection and dissection alternatives. The data was collected through a mixed methods approach involving online surveys (n = 153) and subsequent telephone interviews (n = 9) with secondary school science and biology teachers. The findings indicate…

  1. [Retrograde type A dissection after endovascular stent grafting of type B dissection].

    PubMed

    Misfeld, M; Nötzold, A; Geist, V; Richardt, G; Sievers, H H

    2002-03-01

    Acute aortic dissection is a disease with high mortality. Whereas acute dissection of the ascending aorta (Standford type A) is treated surgically, acute dissection of Stanford type B (descending aorta) is principally treated conservatively, but surgically in case of complications. Recently, another therapeutical option for the treatment of type B dissection has been developed using endovascular stent-grafts. We report on a 64-year-old woman with typical signs of acute aortic dissection. Computer tomography and transesophageal echocardiography demonstrated Stanford type B dissection. The patient was treated with an endovascular stent-graft, because of malperfusion of the right leg and chest pain. After successful closure of the entry by the stent, the patient developed acute right-sided hemiplegia one day after the intervention due to retrograde dissection into the aortic arch and ascending aorta. Upon immediate operation, the origin of the initially type B dissection was still sufficiently occluded by the endovascular stent-graft; however, there was another entry between the innominate artery and the left carotic artery near one proximal end of the stent's strut. Using deep hypothermia and selective antegrade cerebral perfusion, the ascending aorta and proximal arch were replaced with a 28 mm Dacron-Velour tube and the aortic root was remodelled with a tongue-shaped Dacron graft preserving the valve cusps according to a modified Yacoub procedure. After the operation, neurological symptoms diminished and the patient could walk on the ward on day eleven. This case demonstrates retrograde type A dissection as a complication after interventional treatment of type B dissection using an endovascular stent-graft. The reason for this delayed complication is speculative. Aortic wall damage during stent inserting could be a possible cause. It is also likely that the patient initially had type B dissection with retrograde dissection of the distal part of the aortic arch

  2. Genetic dissection of acetic acid tolerance in Saccharomyces cerevisiae.

    PubMed

    Geng, Peng; Xiao, Yin; Hu, Yun; Sun, Haiye; Xue, Wei; Zhang, Liang; Shi, Gui-Yang

    2016-09-01

    Dissection of the hereditary architecture underlying Saccharomyces cerevisiae tolerance to acetic acid is essential for ethanol fermentation. In this work, a genomics approach was used to dissect hereditary variations in acetic acid tolerance between two phenotypically different strains. A total of 160 segregants derived from these two strains were obtained. Phenotypic analysis indicated that the acetic acid tolerance displayed a normal distribution in these segregants, and suggested that the acetic acid tolerant traits were controlled by multiple quantitative trait loci (QTLs). Thus, 220 SSR markers covering the whole genome were used to detect QTLs of acetic acid tolerant traits. As a result, three QTLs were located on chromosomes 9, 12, and 16, respectively, which explained 38.8-65.9 % of the range of phenotypic variation. Furthermore, twelve genes of the candidates fell into the three QTL regions by integrating the QTL analysis with candidates of acetic acid tolerant genes. These results provided a novel avenue to obtain more robust strains. PMID:27430512

  3. Carotid and vertebral artery dissection syndromes

    PubMed Central

    Thanvi, B; Munshi, S; Dawson, S; Robinson, T

    2005-01-01

    Cervicocerebral arterial dissections (CAD) are an important cause of strokes in younger patients accounting for nearly 20% of strokes in patients under the age of 45 years. Extracranial internal carotid artery dissections comprise 70%–80% and extracranial vertebral dissections account for about 15% of all CAD. Aetiopathogenesis of CAD is incompletely understood, though trauma, respiratory infections, and underlying arteriopathy are considered important. A typical picture of local pain, headache, and ipsilateral Horner's syndrome followed after several hours by cerebral or retinal ischaemia is rare. Doppler ultrasound, MRI/MRA, and CT angiography are useful non-invasive diagnostic tests. The treatment of extracranial CAD is mainly medical using anticoagulants or antiplatelet agents although controlled studies to show their effectiveness are lacking. The prognosis of extracranial CAD is generally much better than that of the intracranial CAD. Recurrences are rare in CAD. PMID:15937204

  4. [Medical management of cervical arterial dissections].

    PubMed

    Vuillier, F; Tatu, L; Moulin, T

    2002-12-01

    Medical management of cervical arterial dissections is not standardized and has not been the subject of randomized trials. Management is mainly based on the presumed pathophysiology of secondary cerebral infarcts associated with dissections and the individual experience of each treating team. First, a review of the literature regarding medical management of acute and chronic dissections is presented. Then, results from a national study sponsored by the Société Française Neuro-Vasculaire and the Société Française de Neuro-Radiologie evaluating the medical management of this pathology in French neuro-vascular centers will be presented. These data will be useful to generate practical management recommendations and establish guidelines for further studies.

  5. The nature of dissection: Exploring student conceptions

    NASA Astrophysics Data System (ADS)

    York, Katharine

    The model of conceptual change in science describes the process of learning as a complete restructuring of knowledge, when learners discover or are shown more plausible, intelligent alternatives to existing conceptions. Emotions have been acknowledged as part of a learner's conceptual ecology, but the effects of emotions on learning have yet to be described. This research was conducted to examine the role that emotions have on learning for thirteen high school students, as they dissected cats in a Human Anatomy and Physiology class. The project also investigated whether a student's emotional reactions may be used to develop a sense of connectedness with the nonhuman world, which is defined as ecological literacy. This study utilized a grounded theory approach, in which student responses to interviews were the primary source of data. Interviews were transcribed, and responses were coded according to a constant comparative method of analysis. Responses were compared with the four conditions necessary for conceptual change to occur, and also to five principles of ecological literacy. Students who had negative reactions to dissection participated less in the activity, and demonstrated less conceptual change. Two female students showed the strongest emotional reactions to dissection, and also the lowest amount of conceptual change. One male student also had strong negative reactions to death, and showed no conceptual change. The dissection experiences of the students in this study did not generally reflect ecological principles. The two students whose emotional reactions to dissection were the most negative demonstrated the highest degree of ecological literacy. These results provide empirical evidence of the effects that emotions have on learning, and also supports the opinions of educators who do not favor dissection, because it does not teach students to respect all forms of life.

  6. Intracranial Vertebral Artery Dissections: Evolving Perspectives

    PubMed Central

    Ali, M.S.; Amenta, P.S.; Starke, R.M.; Jabbour, P.M.; Gonzalez, L.F.; Tjoumakaris, S.I.; Flanders, A.E.; Rosenwasser, R.H.; Dumont, A.S.

    2012-01-01

    Summary Intracranial vertebral artery dissection (VAD) represents the underlying etiology in a significant percentage of posterior circulation ischemic strokes and subarachnoid hemorrhages. These lesions are particularly challenging in their diagnosis, management, and in the prediction of long-term outcome. Advances in the understanding of underlying processes leading to dissection, as well as the evolution of modern imaging techniques are discussed. The data pertaining to medical management of intracranial VADs, with emphasis on anticoagulants and antiplatelet agents, is reviewed. Surgical intervention is discussed, including, the selection of operative candidates, open and endovascular procedures, and potential complications. The evolution of endovascular technology and techniques is highlighted. PMID:23217643

  7. Changes in the Phosphoproteome and Metabolome Link Early Signaling Events to Rearrangement of Photosynthesis and Central Metabolism in Salinity and Oxidative Stress Response in Arabidopsis.

    PubMed

    Chen, Yanmei; Hoehenwarter, Wolfgang

    2015-12-01

    Salinity and oxidative stress are major factors affecting and limiting the productivity of agricultural crops. The molecular and biochemical processes governing the plant response to abiotic stress have often been researched in a reductionist manner. Here, we report a systemic approach combining metabolic labeling and phosphoproteomics to capture early signaling events with quantitative metabolome analysis and enzyme activity assays to determine the effects of salt and oxidative stress on plant physiology. K(+) and Na(+) transporters showed coordinated changes in their phosphorylation pattern, indicating the importance of dynamic ion homeostasis for adaptation to salt stress. Unique phosphorylation sites were found for Arabidopsis (Arabidopsis thaliana) SNF1 kinase homolog10 and 11, indicating their central roles in the stress-regulated responses. Seven Sucrose Non-fermenting1-Related Protein Kinase2 kinases showed varying levels of phosphorylation at multiple serine/threonine residues in their kinase domain upon stress, showing temporally distinct modulation of the various isoforms. Salinity and oxidative stress also lead to changes in protein phosphorylation of proteins central to photosynthesis, in particular the kinase State Transition Protein7 required for state transition and light-harvesting II complex proteins. Furthermore, stress-induced changes of the phosphorylation of enzymes of central metabolism were observed. The phosphorylation patterns of these proteins were concurrent with changes in enzyme activity. This was reflected by altered levels of metabolites, such as the sugars sucrose and fructose, glycolysis intermediates, and amino acids. Together, our study provides evidence for a link between early signaling in the salt and oxidative stress response that regulates the state transition of photosynthesis and the rearrangement of primary metabolism.

  8. Niobium(V) oxide (Nb2O5): application to phosphoproteomics.

    PubMed

    Ficarro, Scott B; Parikh, Jignesh R; Blank, Nathaniel C; Marto, Jarrod A

    2008-06-15

    Proteomics-based analysis of signaling cascades relies on a growing suite of affinity resins and methods aimed at efficient enrichment of phosphorylated peptides from complex biological mixtures. Given the heterogeneity of phosphopeptides and the overlap in chemical properties between phospho- and unmodified peptides, it is likely that the use of multiple resins will provide the best combination of specificity, yield, and coverage for large-scale proteomics studies. Recently titanium and zirconium dioxides have been used successfully for enrichment of phosphopeptides. Here we report the first demonstration that niobium pentoxide (Nb 2O 5) provides for efficient enrichment and recovery ( approximately 50-100%) of phosphopeptides from simple mixtures and facilitates identification of several hundred putative sites of phosphorylation from cell lysate. Comparison of phosphorylated peptides identified from Nb 2O 5 and TiO 2 with sequences in the PhosphoELM database suggests a useful degree of divergence in the selectivity of these metal oxide resins. Collectively our data indicate that Nb 2O 5 provides efficient enrichment for phosphopeptides and offers a complementary approach for large-scale phosphoproteomics studies.

  9. Visualization and Biochemical Analyses of the Emerging Mammalian 14-3-3-Phosphoproteome*

    PubMed Central

    Johnson, Catherine; Tinti, Michele; Wood, Nicola T.; Campbell, David G.; Toth, Rachel; Dubois, Fanny; Geraghty, Kathryn M.; Wong, Barry H. C.; Brown, Laura J.; Tyler, Jennifer; Gernez, Aurélie; Chen, Shuai; Synowsky, Silvia; MacKintosh, Carol

    2011-01-01

    Hundreds of candidate 14-3-3-binding (phospho)proteins have been reported in publications that describe one interaction at a time, as well as high-throughput 14-3-3-affinity and mass spectrometry-based studies. Here, we transcribed these data into a common format, deposited the collated data from low-throughput studies in MINT (http://mint.bio.uniroma2.it/mint), and compared the low- and high-throughput data in VisANT graphs that are easy to analyze and extend. Exploring the graphs prompted questions about technical and biological specificity, which were addressed experimentally, resulting in identification of phosphorylated 14-3-3-binding sites in the mitochondrial import sequence of the iron-sulfur cluster assembly enzyme (ISCU), cytoplasmic domains of the mitochondrial fission factor (MFF), and endoplasmic reticulum-tethered receptor expression-enhancing protein 4 (REEP4), RNA regulator SMAUG2, and cytoskeletal regulatory proteins, namely debrin-like protein (DBNL) and kinesin light chain (KLC) isoforms. Therefore, 14-3-3s undergo physiological interactions with proteins that are destined for diverse subcellular locations. Graphing and validating interactions underpins efforts to use 14-3-3-phosphoproteomics to identify mechanisms and biomarkers for signaling pathways in health and disease. PMID:21725060

  10. The phosphoproteome of Aspergillus nidulans reveals functional association with cellular processes involved in morphology and secretion.

    PubMed

    Ramsubramaniam, Nikhil; Harris, Steven D; Marten, Mark R

    2014-11-01

    We describe the first phosphoproteome of the model filamentous fungus Aspergillus nidulans. Phosphopeptides were enriched using titanium dioxide, separated using a convenient ultra-long reverse phase gradient, and identified using a "high-high" strategy (high mass accuracy on the parent and fragment ions) with higher-energy collisional dissociation. Using this approach 1801 phosphosites, from 1637 unique phosphopeptides, were identified. Functional classification revealed phosphoproteins were overrepresented under GO categories related to fungal morphogenesis: "sites of polar growth," "vesicle mediated transport," and "cytoskeleton organization." In these same GO categories, kinase-substrate analysis of phosphoproteins revealed the majority were target substrates of CDK and CK2 kinase families, indicating these kinase families play a prominent role in fungal morphogenesis. Kinase-substrate analysis also identified 57 substrates for kinases known to regulate secretion of hydrolytic enzymes (e.g. PkaA, SchA, and An-Snf1). Altogether this data will serve as a benchmark that can be used to elucidate regulatory networks functionally associated with fungal morphogenesis and secretion. All MS data have been deposited in the ProteomeXchange with identifier PXD000715 (http://proteomecentral.proteomexchange.org/dataset/PXD000715).

  11. The proteome and phosphoproteome of Neurospora crassa in response to cellulose, sucrose and carbon starvation

    PubMed Central

    Xiong, Yi; Coradetti, Samuel T.; Li, Xin; Gritsenko, Marina A.; Clauss, Therese; Petyuk, Vlad; Camp, David; Smith, Richard; Cate, Jamie H.D.; Yang, Feng; Glass, N. Louise

    2014-01-01

    Improving cellulolytic enzyme production by plant biomass degrading fungi holds great potential in reducing costs associated with production of next-generation biofuels generated from lignocellulose. How fungi sense cellulosic materials and respond by secreting enzymes has mainly been examined by assessing function of transcriptional regulators and via transcriptional profiling. Here, we obtained global proteomic and phosphoproteomic profiles of the plant biomass degrading filamentous fungus Neurospora crassa grown on different carbon sources, i.e. sucrose, no carbon, and cellulose, by performing isobaric tags for relative and absolute quantification (iTRAQ)-based LC–MS/MS analyses. A comparison between proteomes and transcriptomes under identical carbon conditions suggests that extensive post-transcriptional regulation occurs in N. crassa in response to exposure to cellulosic material. Several hundred amino acid residues with differential phosphorylation levels on crystalline cellulose (Avicel) or carbon-free medium vs sucrose medium were identified, including phosphorylation sites in a major transcriptional activator for cellulase genes, CLR1, as well as a cellobionic acid transporter, CBT1. Mutation of phosphorylation sites on CLR1 did not have a major effect on transactivation of cellulase production, while mutation of phosphorylation sites in CBT1 increased its transporting capacity. Our data provides rich information at both the protein and phosphorylation levels of the early cellular responses to carbon starvation and cellulosic induction and aids in a greater understanding of the underlying post-transcriptional regulatory mechanisms in filamentous fungi. PMID:24881580

  12. The proteome and phosphoproteome of Neurospora crassa in response to cellulose, sucrose and carbon starvation

    SciTech Connect

    Xiong, Yi; Coradetti, Samuel T.; Li, Xin; Gritsenko, Marina A.; Clauss, Therese; Petyuk, Vlad; Camp, David; Smith, Richard; Cate, Jamie H. D.; Yang, Feng; Glass, N. Louise

    2014-05-29

    Improving cellulolytic enzyme production by plant biomass degrading fungi holds great potential in reducing costs associated with production of next-generation biofuels generated from lignocellulose. How fungi sense cellulosic materials and respond by secreting enzymes has mainly been examined by assessing function of transcriptional regulators and via transcriptional profiling. Here, we obtained global proteomic and phosphoproteomic profiles of the plant biomass degrading filamentous fungus Neurospora crassa grown on different carbon sources, i.e. sucrose, no carbon, and cellulose, by performing isobaric tags for relative and absolute quantification (iTRAQ) -based LC-MS/MS analyses. A comparison between proteomes and transcriptomes under identical carbon conditions suggests that extensive post-transcriptional regulation occurs in N. crassa in response to exposure to cellulosic material. Several hundred amino acid residues with differential phosphorylation levels on crystalline cellulose (Avicel) or carbon-free medium versus sucrose medium were identified, including phosphorylation sites in a major transcriptional activator for cellulase genes, CLR1, as well as a cellobionic acid transporter, CBT1. Finally, we found mutation of phosphorylation sites on CLR1 did not have a major effect on transactivation of cellulase production, while mutation of phosphorylation sites in CBT1 increased its transporting capacity. Our data provides rich information at both the protein and phosphorylation levels of the early cellular responses to carbon starvation and cellulosic induction and aids in a greater understanding of the underlying post-transcriptional regulatory mechanisms in filamentous fungi.

  13. The proteome and phosphoproteome of Neurospora crassa in response to cellulose, sucrose and carbon starvation

    DOE PAGESBeta

    Xiong, Yi; Coradetti, Samuel T.; Li, Xin; Gritsenko, Marina A.; Clauss, Therese; Petyuk, Vlad; Camp, David; Smith, Richard; Cate, Jamie H. D.; Yang, Feng; et al

    2014-05-29

    Improving cellulolytic enzyme production by plant biomass degrading fungi holds great potential in reducing costs associated with production of next-generation biofuels generated from lignocellulose. How fungi sense cellulosic materials and respond by secreting enzymes has mainly been examined by assessing function of transcriptional regulators and via transcriptional profiling. Here, we obtained global proteomic and phosphoproteomic profiles of the plant biomass degrading filamentous fungus Neurospora crassa grown on different carbon sources, i.e. sucrose, no carbon, and cellulose, by performing isobaric tags for relative and absolute quantification (iTRAQ) -based LC-MS/MS analyses. A comparison between proteomes and transcriptomes under identical carbon conditions suggestsmore » that extensive post-transcriptional regulation occurs in N. crassa in response to exposure to cellulosic material. Several hundred amino acid residues with differential phosphorylation levels on crystalline cellulose (Avicel) or carbon-free medium versus sucrose medium were identified, including phosphorylation sites in a major transcriptional activator for cellulase genes, CLR1, as well as a cellobionic acid transporter, CBT1. Finally, we found mutation of phosphorylation sites on CLR1 did not have a major effect on transactivation of cellulase production, while mutation of phosphorylation sites in CBT1 increased its transporting capacity. Our data provides rich information at both the protein and phosphorylation levels of the early cellular responses to carbon starvation and cellulosic induction and aids in a greater understanding of the underlying post-transcriptional regulatory mechanisms in filamentous fungi.« less

  14. Phosphoproteome and Transcriptome of RA-Responsive and RA-Resistant Breast Cancer Cell Lines.

    PubMed

    Carrier, Marilyn; Joint, Mathilde; Lutzing, Régis; Page, Adeline; Rochette-Egly, Cécile

    2016-01-01

    Retinoic acid (RA), the main active vitamin A metabolite, controls multiple biological processes such as cell proliferation and differentiation through genomic programs and kinase cascades activation. Due to these properties, RA has proven anti-cancer capacity. Several breast cancer cells respond to the antiproliferative effects of RA, while others are RA-resistant. However, the overall signaling and transcriptional pathways that are altered in such cells have not been elucidated. Here, in a large-scale analysis of the phosphoproteins and in a genome-wide analysis of the RA-regulated genes, we compared two human breast cancer cell lines, a RA-responsive one, the MCF7 cell line, and a RA-resistant one, the BT474 cell line, which depicts several alterations of the "kinome". Using high-resolution nano-LC-LTQ-Orbitrap mass spectrometry associated to phosphopeptide enrichment, we found that several proteins involved in signaling and in transcription, are differentially phosphorylated before and after RA addition. The paradigm of these proteins is the RA receptor α (RARα), which was phosphorylated in MCF7 cells but not in BT474 cells after RA addition. The panel of the RA-regulated genes was also different. Overall our results indicate that RA resistance might correlate with the deregulation of the phosphoproteome with consequences on gene expression.

  15. Phosphoproteome and Transcriptome of RA-Responsive and RA-Resistant Breast Cancer Cell Lines

    PubMed Central

    Carrier, Marilyn; Joint, Mathilde; Lutzing, Régis; Page, Adeline; Rochette-Egly, Cécile

    2016-01-01

    Retinoic acid (RA), the main active vitamin A metabolite, controls multiple biological processes such as cell proliferation and differentiation through genomic programs and kinase cascades activation. Due to these properties, RA has proven anti-cancer capacity. Several breast cancer cells respond to the antiproliferative effects of RA, while others are RA-resistant. However, the overall signaling and transcriptional pathways that are altered in such cells have not been elucidated. Here, in a large-scale analysis of the phosphoproteins and in a genome-wide analysis of the RA-regulated genes, we compared two human breast cancer cell lines, a RA-responsive one, the MCF7 cell line, and a RA-resistant one, the BT474 cell line, which depicts several alterations of the “kinome”. Using high-resolution nano-LC-LTQ-Orbitrap mass spectrometry associated to phosphopeptide enrichment, we found that several proteins involved in signaling and in transcription, are differentially phosphorylated before and after RA addition. The paradigm of these proteins is the RA receptor α (RARα), which was phosphorylated in MCF7 cells but not in BT474 cells after RA addition. The panel of the RA-regulated genes was also different. Overall our results indicate that RA resistance might correlate with the deregulation of the phosphoproteome with consequences on gene expression. PMID:27362937

  16. Phosphoproteomic dynamics of chickpea (Cicer arietinum L.) reveals shared and distinct components of dehydration response.

    PubMed

    Subba, Pratigya; Barua, Pragya; Kumar, Rajiv; Datta, Asis; Soni, Kamlesh Kumar; Chakraborty, Subhra; Chakraborty, Niranjan

    2013-11-01

    Reversible protein phosphorylation is a ubiquitous regulatory mechanism that plays critical roles in transducing stress signals to bring about coordinated intracellular responses. To gain better understanding of dehydration response in plants, we have developed a differential phosphoproteome in a food legume, chickpea (Cicer arietinum L.). Three-week-old chickpea seedlings were subjected to progressive dehydration by withdrawing water, and the changes in the phosphorylation status of a large repertoire of proteins were monitored. The proteins were resolved by 2-DE and stained with phosphospecific fluorescent Pro-Q Diamond dye. Mass spectrometric analysis led to the identification of 91 putative phosphoproteins, presumably involved in a variety of functions including cell defense and rescue, photosynthesis and photorespiration, molecular chaperones, and ion transport, among others. Multiple sites of phosphorylation were predicted on several key elements, which include both the regulatory as well as the functional proteins. A critical survey of the phosphorylome revealed a DREPP (developmentally regulated plasma membrane protein) plasma membrane polypeptide family protein, henceforth designated CaDREPP1. The transcripts of CaDREPP1 were found to be differentially regulated under dehydration stress, further corroborating the proteomic results. This work provides new insights into the possible phosphorylation events triggered by the conditions of progressive water-deficit in plants.

  17. Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases

    PubMed Central

    Steger, Martin; Tonelli, Francesca; Ito, Genta; Davies, Paul; Trost, Matthias; Vetter, Melanie; Wachter, Stefanie; Lorentzen, Esben; Duddy, Graham; Wilson, Stephen; Baptista, Marco AS; Fiske, Brian K; Fell, Matthew J; Morrow, John A; Reith, Alastair D; Alessi, Dario R; Mann, Matthias

    2016-01-01

    Mutations in Park8, encoding for the multidomain Leucine-rich repeat kinase 2 (LRRK2) protein, comprise the predominant genetic cause of Parkinson's disease (PD). G2019S, the most common amino acid substitution activates the kinase two- to threefold. This has motivated the development of LRRK2 kinase inhibitors; however, poor consensus on physiological LRRK2 substrates has hampered clinical development of such therapeutics. We employ a combination of phosphoproteomics, genetics, and pharmacology to unambiguously identify a subset of Rab GTPases as key LRRK2 substrates. LRRK2 directly phosphorylates these both in vivo and in vitro on an evolutionary conserved residue in the switch II domain. Pathogenic LRRK2 variants mapping to different functional domains increase phosphorylation of Rabs and this strongly decreases their affinity to regulatory proteins including Rab GDP dissociation inhibitors (GDIs). Our findings uncover a key class of bona-fide LRRK2 substrates and a novel regulatory mechanism of Rabs that connects them to PD. DOI: http://dx.doi.org/10.7554/eLife.12813.001 PMID:26824392

  18. The differential hippocampal phosphoproteome of Apodemus sylvaticus paralleling spatial memory retrieval in the Barnes maze.

    PubMed

    Li, Lin; Csaszar, Edina; Szodorai, Edit; Patil, Sudarshan; Pollak, Arnold; Lubec, Gert

    2014-05-01

    Protein phosphorylation is a well-known and well-documented mechanism in memory processes. Although a large series of protein kinases involved in memory processes have been reported, information on phosphoproteins is limited. It was therefore the aim of the study to determine a partial and differential phosphoproteome along with the corresponding network in hippocampus of a wild caught mouse strain with excellent performance in several paradigms of spatial memory. Apodemus sylvaticus mice were trained in the Barnes maze, a non-invasive test system for spatial memory and untrained mice served as controls. Animals were sacrificed 6h following memory retrieval, hippocampi were taken, proteins extracted and in-solution digestion was carried out with subsequent iTRAQ double labelling. Phosphopeptides were enriched by a TiO2-based method and semi-quantified using two fragmentation principles on the LTQ-orbitrap Velos. In hippocampi of trained animals phosphopeptide levels representing signalling, neuronal, synaptosomal, cytoskeletal and metabolism proteins were at least twofold reduced or increased. Furthermore, a network revealing a link to pathways of ubiquitination, the androgen receptor, small GTPase Rab5 and MAPK signaling as well as synucleins was constructed. This work is relevant for interpretation of previous work and the design of future studies on protein phosphorylation in spatial memory.

  19. The phosphoproteome of Arabidopsis plants lacking the oxidative signal-inducible1 (OXI1) protein kinase.

    PubMed

    Howden, Andrew J M; Salek, Mogjiborahman; Miguet, Laurent; Pullen, Margaret; Thomas, Benjamin; Knight, Marc R; Sweetlove, Lee J

    2011-04-01

    The AGC protein kinase OXI1 is a key protein in plant responses to oxidative signals, and is important for two oxidative burst-mediated processes: basal resistance to microbial pathogens and root hair growth. To identify possible components of the OXI1 signalling pathway, phosphoproteomic techniques were used to detect alterations in the abundance of phosphorylated proteins and peptides in an oxi1 null mutant of Arabidopsis thaliana. The relative abundance of phosphorylated proteins was assessed either using two-dimensional gel electrophoresis and staining with the phosphoprotein stain Pro-Q Diamond or by the identification and quantification, by mass spectrometry, of stable-isotope labelled phosphopeptides. A number of proteins show altered phosphorylation in the oxi1 mutant. Five proteins, including a putative F-box and 3-phosphoinositide-dependent kinase 1, show reduced phosphorylation in the oxi1 mutant, and may be direct or indirect targets of OXI1. Four proteins, including ethylene insensitive 2 and phospholipase d-gamma, show increased phosphorylation in the oxi1 mutant. This study has identified a range of candidate proteins from the OXI1 signalling pathway. The diverse activities of these proteins, including protein degradation and hormone signalling, may suggest crosstalk between OXI1 and other signal transduction cascades.

  20. Human cytomegalovirus pUL97 kinase induces global changes in the infected cell phosphoproteome

    PubMed Central

    Oberstein, Adam; Perlman, David H.; Shenk, Thomas; Terry, Laura J.

    2015-01-01

    Replication of human cytomegalovirus is regulated in part by cellular kinases and the single viral Ser/Thr kinase, pUL97. The virus-coded kinase augments the replication of human cytomegalovirus (HCMV) by enabling nuclear egress and altering cell cycle progression. These roles are accomplished through direct phosphorylation of nuclear lamins and the retinoblastoma protein, respectively. In an effort to identify additional pUL97 substrates, we analyzed the phosphoproteome of SILAC-labeled human fibroblasts during infection with either wild-type HCMV or a pUL97 kinase-dead mutant virus. Phosphopeptides were enriched over a titanium dioxide matrix and analyzed by high resolution mass spectrometry. We identified 157 unambiguous phosphosites from 106 cellular and 17 viral proteins whose phosphorylation required UL97. Analysis of peptides containing these sites allowed the identification of several candidate pUL97 phosphorylation motifs, including a completely novel phosphorylation motif, LxSP. Substrates harboring the LxSP motif were enriched in nucleocytoplasmic transport functions, including a number of components of the nuclear pore complex. These results extend the known functions of pUL97 and suggest that modulation of nuclear pore function may be important during HCMV replication. PMID:25867546

  1. Conservative Management of Chronic Aortic Dissection with Underlying Aortic Aneurysm

    PubMed Central

    Yusuf Beebeejaun, Mohammad; Malec, Aleksandra; Gupta, Ravi

    2013-01-01

    Aortic dissection is one of the most common aortic emergencies affecting around 2000 Americans each year. It usually presents in the acute state but in a small percentage of patients aortic dissections go unnoticed and these patients survive without any adequate therapy. With recent advances in medical care and diagnostic technologies, aortic dissection can be successfully managed through surgical or medical options, consequently increasing the related survival rate. However, little is known about the optimal long-term management of patients suffering from chronic aortic dissection. The purpose of the present report is to review aortic dissection, namely its pathology and the current diagnostic tools available, and to discuss the management options for chronic aortic dissection. We report a patient in which chronic aortic dissection presented with recurring episodes of vomiting and also discuss the management plan of our patient who had a chronic aortic dissection as well as an underlying aortic aneurysm. PMID:24179638

  2. Genetics Home Reference: familial thoracic aortic aneurysm and dissection

    MedlinePlus

    ... Home Health Conditions familial TAAD familial thoracic aortic aneurysm and dissection Enable Javascript to view the expand/ ... Open All Close All Description Familial thoracic aortic aneurysm and dissection ( familial TAAD ) involves problems with the ...

  3. Squid Dissection: From Pen to Ink.

    ERIC Educational Resources Information Center

    Brown, Cindy; Kisiel, Jim

    2003-01-01

    Introduces students to dissection, which is an important part of scientific discovery. Students not only gain an understanding of the anatomy of a squid, but also develop a sense of responsibility and respect for the animal that they are using as a learning tool. (Author/SOE)

  4. Cow's Eye Dissection in the Physics Lab.

    ERIC Educational Resources Information Center

    Lapp, David R.; Keenan, James E.

    1991-01-01

    Proposes the science demonstration of dissecting a cow's eye to integrate biology and physics in the study of optics and lenses. Reviews the anatomy of the eye, describes the visual process and covers topics as index of refraction of the cornea, microscopic receptors, the lens, and the retina. (MDH)

  5. College Students' Opinions about Animal Dissections.

    ERIC Educational Resources Information Center

    Lord, Thomas; Moses, Richard

    1994-01-01

    Provides brief arguments for and against the use of animals in research and education. A survey was conducted of 200 students to seek answers to the following questions: How do students feel about the dissection of animals in life science classes?; and Do students feel they would learn as much biology without laboratory animal investigations?…

  6. Intraoperative aortic dissection in pediatric heart surgery.

    PubMed

    Hibino, Narutoshi; Harada, Yorikazu; Hiramatsu, Takeshi; Yasukochi, Satoshi; Satomi, Gengi

    2006-06-01

    Intraoperative aortic dissection occurred in a 3-year-old-boy undergoing repair of an atrial septal defect. Transesophageal echocardiography was useful for the diagnosis, and conservative medical treatment under close observation was feasible in this case which involved a limited intimal tear. PMID:16714685

  7. Renal infarction resulting from traumatic renal artery dissection.

    PubMed

    Kang, Kyung Pyo; Lee, Sik; Kim, Won; Jin, Gong Yong; Na, Ki Ryang; Yun, Il Yong; Park, Sung Kwang

    2008-06-01

    Renal artery dissection may be caused by iatrogenic injury, trauma, underlying arterial diseases such as fibromuscular disease, atherosclerotic disease, or connective tissue disease. Radiological imaging may be helpful in detecting renal artery pathology, such as renal artery dissection. For patients with acute, isolated renal artery dissection, surgical treatment, endovascular management, or medical treatment have been considered effective measures to preserve renal function. We report a case of renal infarction that came about as a consequence of renal artery dissection.

  8. [Surgical aspects of acute aortic dissection].

    PubMed

    Laas, J; Heinemann, M; Jurmann, M; Borst, H G

    1992-12-01

    This paper highlights some of the surgical aspects of acute aortic dissections such as: emergency diagnosis, indications for surgery, reconstructive operative techniques, malperfusion phenomena and necessity for follow-up. Aortic dissection is caused by an intimal tear, called the "entry", and subsequent splitting of the media by the stream of blood. Two lumina are thus created, which may communicate through "re-entries". As this creates severe weakness of the aortic wall, rupture and/or dilatation are the imminent dangers of acute aortic dissection. Acute aortic dissection type A, by definition involving the ascending aorta (Figures 1 and 2), is an absolute indication for emergency surgical treatment, because its natural history shows an extremely poor outcome (Figure 3). Due to impending (intrapericardial) aortic rupture, it may be necessary to limit diagnostic procedures to a minimum. Transesophageal echocardiography is the method of choice for establishing a quick, precise and reliable diagnosis (Figure 4). In stable patients, computed tomography gives additional information about aortic diameters or sites of extrapericardial perforation. Digital subtraction angiography (DSA) shows perfusion of the lumina and dependent organs. The surgical strategy in acute aortic dissection type A aims at replacement of the ascending aorta. Reconstructive techniques have to be considered, especially in aortic valve regurgitation without annuloectasia (Figures 5 and 6). In recent times, the use of GRF tissue glue has reduced the need for teflon felt. Involvement of the aortic arch should be treated aggressively up to the point of total arch replacement in deep hypothermic circulatory arrest as part of the primary procedure (Figure 7). Malperfusion phenomena of aortic branches remain risk-factors.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Preservation of the optic radiations based on comparative analysis of diffusion tensor imaging tractography and anatomical dissection

    PubMed Central

    Nooij, Roland P.; Hoving, Eelco W.; van Hulzen, Arjen L. J.; Cornelissen, Frans W.; Renken, Remco J.

    2015-01-01

    Background: Visualization of the precise course of the visual pathways is relevant to prevent damage that may inflict visual field deficits during neurosurgical resections. In particular the optic radiations (OR) are susceptible to such damage during neurosurgery. Cortical pathways can be mapped in vivo, by using Diffusion Tensor Imaging (DTI). Visualization of these pathways would be potentially helpful to prevent neurosurgical visual morbidity. In this study an anatomical dissection of the visual pathways was compared to DTI fiber tractography (DTI-FT) data of four human brains. The feasibility of a definition of a Safety Zone is investigated. Methods: Four adult brains were dissected using Klingler's fiber dissection method, which allowed preparation of the OR. Measurements before and after dissection were used to establish distances from the cortex to the OR. DTI-scans were also obtained from these brains to determine the same distances. Results: Measurements from specific landmark points on the cortex to the lateral border of the OR were performed in four brains. Analysis through DTI tractography corresponded with the dissection results. Based on the combined results of both dissection and DTI-FT, we defined a quantitative surgical Safety Zone with respect to various anatomical landmarks (in particular the ventricle system). Conclusion: We conclude that there is a good correlation between the visualizations of the optic pathways based on dissection and DTI. Furthermore, we conclude that defining a neurosurgical Safety Zone which could preserve the integrity of the OR during surgery, based on the combination of DTI-FT images and dissection is feasible. PMID:26300739

  10. iPhos: a toolkit to streamline the alkaline phosphatase-assisted comprehensive LC-MS phosphoproteome investigation

    PubMed Central

    2014-01-01

    Background Comprehensive characterization of the phosphoproteome in living cells is critical in signal transduction research. But the low abundance of phosphopeptides among the total proteome in cells remains an obstacle in mass spectrometry-based proteomic analysis. To provide a solution, an alternative analytic strategy to confidently identify phosphorylated peptides by using the alkaline phosphatase (AP) treatment combined with high-resolution mass spectrometry was provided. While the process is applicable, the key integration along the pipeline was mostly done by tedious manual work. Results We developed a software toolkit, iPhos, to facilitate and streamline the work-flow of AP-assisted phosphoproteome characterization. The iPhos tookit includes one assister and three modules. The iPhos Peak Extraction Assister automates the batch mode peak extraction for multiple liquid chromatography mass spectrometry (LC-MS) runs. iPhos Module-1 can process the peak lists extracted from the LC-MS analyses derived from the original and dephosphorylated samples to mine out potential phosphorylated peptide signals based on mass shift caused by the loss of some multiples of phosphate groups. And iPhos Module-2 provides customized inclusion lists with peak retention time windows for subsequent targeted LC-MS/MS experiments. Finally, iPhos Module-3 facilitates to link the peptide identifications from protein search engines to the quantification results from pattern-based label-free quantification tools. We further demonstrated the utility of the iPhos toolkit on the data of human metastatic lung cancer cells (CL1-5). Conclusions In the comparison study of the control group of CL1-5 cell lysates and the treatment group of dasatinib-treated CL1-5 cell lysates, we demonstrated the applicability of the iPhos toolkit and reported the experimental results based on the iPhos-facilitated phosphoproteome investigation. And further, we also compared the strategy with pure DDA-based LC

  11. Which Comes First: The Use of Computer Simulation of Frog Dissection or Conventional Dissection as Academic Exercise?

    ERIC Educational Resources Information Center

    Akpan, Joseph; Strayer, Jeremy

    2010-01-01

    Science educators and school administrators are reexamining the educational value of animal dissection in the nation's schools and are focusing on simulation as an instructional alternative. One implication of the debate is that simulations can lead to equivalent learning to hands-on dissection. The second implication is whether dissection is…

  12. Immobilized metal affinity chromatography on collapsed Langmuir-Blodgett iron(III) stearate films and iron(III) oxide nanoparticles for bottom-up phosphoproteomics.

    PubMed

    Gladilovich, Vladimir; Greifenhagen, Uta; Sukhodolov, Nikolai; Selyutin, Artem; Singer, David; Thieme, Domenika; Majovsky, Petra; Shirkin, Alexey; Hoehenwarter, Wolfgang; Bonitenko, Evgeny; Podolskaya, Ekaterina; Frolov, Andrej

    2016-04-22

    Phosphorylation is the enzymatic reaction of site-specific phosphate transfer from energy-rich donors to the side chains of serine, threonine, tyrosine, and histidine residues in proteins. In living cells, reversible phosphorylation underlies a universal mechanism of intracellular signal transduction. In this context, analysis of the phosphoproteome is a prerequisite to better understand the cellular regulatory networks. Conventionally, due to the low contents of signaling proteins, selective enrichment of proteolytic phosphopeptides by immobilized metal affinity chromatography (IMAC) is performed prior to their LC-MS or -MS/MS analysis. Unfortunately, this technique still suffers from low selectivity and compromised analyte recoveries. To overcome these limitations, we propose IMAC systems comprising stationary phases based on collapsed Langmuir-Blodgett films of iron(III) stearate (FF) or iron(III) oxide nanoparticles (FO) and mobile phases relying on ammonia, piperidine and heptadecafluorooctanesulfonic acid (PFOS). Experiments with model phosphopeptides and phosphoprotein tryptic digests showed superior binding capacity, selectivity and recovery for both systems in comparison to the existing commercial analogs. As evidenced by LC-MS/MS analysis of the HeLa phosphoproteome, these features of the phases resulted in increased phosphoproteome coverage in comparison to the analogous commercially available phases, indicating that our IMAC protocol is a promising chromatographic tool for in-depth phosphoproteomic research.

  13. Immobilized metal affinity chromatography on collapsed Langmuir-Blodgett iron(III) stearate films and iron(III) oxide nanoparticles for bottom-up phosphoproteomics.

    PubMed

    Gladilovich, Vladimir; Greifenhagen, Uta; Sukhodolov, Nikolai; Selyutin, Artem; Singer, David; Thieme, Domenika; Majovsky, Petra; Shirkin, Alexey; Hoehenwarter, Wolfgang; Bonitenko, Evgeny; Podolskaya, Ekaterina; Frolov, Andrej

    2016-04-22

    Phosphorylation is the enzymatic reaction of site-specific phosphate transfer from energy-rich donors to the side chains of serine, threonine, tyrosine, and histidine residues in proteins. In living cells, reversible phosphorylation underlies a universal mechanism of intracellular signal transduction. In this context, analysis of the phosphoproteome is a prerequisite to better understand the cellular regulatory networks. Conventionally, due to the low contents of signaling proteins, selective enrichment of proteolytic phosphopeptides by immobilized metal affinity chromatography (IMAC) is performed prior to their LC-MS or -MS/MS analysis. Unfortunately, this technique still suffers from low selectivity and compromised analyte recoveries. To overcome these limitations, we propose IMAC systems comprising stationary phases based on collapsed Langmuir-Blodgett films of iron(III) stearate (FF) or iron(III) oxide nanoparticles (FO) and mobile phases relying on ammonia, piperidine and heptadecafluorooctanesulfonic acid (PFOS). Experiments with model phosphopeptides and phosphoprotein tryptic digests showed superior binding capacity, selectivity and recovery for both systems in comparison to the existing commercial analogs. As evidenced by LC-MS/MS analysis of the HeLa phosphoproteome, these features of the phases resulted in increased phosphoproteome coverage in comparison to the analogous commercially available phases, indicating that our IMAC protocol is a promising chromatographic tool for in-depth phosphoproteomic research. PMID:27016113

  14. Multidimensional strategy for sensitive phosphoproteomics incorporating protein prefractionation combined with SIMAC, HILIC, and TiO(2) chromatography applied to proximal EGF signaling.

    PubMed

    Engholm-Keller, Kasper; Hansen, Thomas Aarup; Palmisano, Giuseppe; Larsen, Martin R

    2011-12-01

    Comprehensive enrichment and fractionation is essential to obtain a broad coverage of the phosphoproteome. This inevitably leads to sample loss, and thus, phosphoproteomics studies are usually only performed on highly abundant samples. Here, we present a comprehensive phosphoproteomics strategy applied to 400 μg of protein from EGF-stimulated HeLa cells. The proteins are separated into membrane and cytoplasmic fractions using sodium carbonate combined with ultracentrifugation. The phosphopeptides were separated into monophosphorylated and multiphosphorylated pools using sequential elution from IMAC (SIMAC) followed by hydrophilic interaction liquid chromatography of the mono- and nonphosphorylated peptides and subsequent titanium dioxide chromatography of the HILIC fractions. This strategy facilitated the identification of >4700 unique phosphopeptides, while 636 phosphosites were changing following short-term EGF stimulation, many of which were not previously known to be involved in EGFR signaling. We further compared three different data processing programs and found large differences in their peptide identification rates due to different implementations of recalibration and filtering. Manually validating a subset of low-scoring peptides exclusively identified using the MaxQuant software revealed a large percentage of false positive identifications. This indicates that, despite having highly accurate precursor mass determination, peptides with low fragment ion scores should not automatically be reported in phosphoproteomics studies. PMID:21955146

  15. Salt-induced changes in cardiac phosphoproteome in a rat model of chronic renal failure.

    PubMed

    Su, Zhengxiu; Zhu, Hongguo; Zhang, Menghuan; Wang, Liangliang; He, Hanchang; Jiang, Shaoling; Hou, Fan Fan; Li, Aiqing

    2014-01-01

    Heart damage is widely present in patients with chronic kidney disease. Salt diet is the most important environmental factor affecting development of chronic renal failure and cardiovascular diseases. The proteins involved in chronic kidney disease -induced heart damage, especially their posttranslational modifications, remain largely unknown to date. Sprague-Dawley rats underwent 5/6 nephrectomy (chronic renal failure model) or sham operation were treated for 2 weeks with a normal-(0.4% NaCl), or high-salt (4% NaCl) diet. We employed TiO2 enrichment, iTRAQ labeling and liquid-chromatography tandem mass spectrometry strategy for phosphoproteomic profiling of left ventricular free walls in these animals. A total of 1724 unique phosphopeptides representing 2551 non-redundant phosphorylation sites corresponding to 763 phosphoproteins were identified. During normal salt feeding, 89 (54%) phosphopeptides upregulated and 76 (46%) phosphopeptides downregulated in chronic renal failure rats relative to sham rats. In chronic renal failure rats, high salt intake induced upregulation of 84 (49%) phosphopeptides and downregulation of 88 (51%) phosphopeptides. Database searches revealed that most of the identified phospholproteins were important signaling molecules such as protein kinases, receptors and phosphatases. These phospholproteins were involved in energy metabolism, cell communication, cell differentiation, cell death and other biological processes. The Search Tool for the Retrieval of Interacting Genes analysis revealed functional links among 15 significantly regulated phosphoproteins in chronic renal failure rats compared to sham group, and 23 altered phosphoproteins induced by high salt intake. The altered phosphorylation levels of two proteins involved in heart damage, lamin A and phospholamban were validated. Expression of the downstream genes of these two proteins, desmin and SERCA2a, were also analyzed.

  16. Identifying drug effects via pathway alterations using an integer linear programming optimization formulation on phosphoproteomic data.

    PubMed

    Mitsos, Alexander; Melas, Ioannis N; Siminelakis, Paraskeuas; Chairakaki, Aikaterini D; Saez-Rodriguez, Julio; Alexopoulos, Leonidas G

    2009-12-01

    Understanding the mechanisms of cell function and drug action is a major endeavor in the pharmaceutical industry. Drug effects are governed by the intrinsic properties of the drug (i.e., selectivity and potency) and the specific signaling transduction network of the host (i.e., normal vs. diseased cells). Here, we describe an unbiased, phosphoproteomic-based approach to identify drug effects by monitoring drug-induced topology alterations. With our proposed method, drug effects are investigated under diverse stimulations of the signaling network. Starting with a generic pathway made of logical gates, we build a cell-type specific map by constraining it to fit 13 key phopshoprotein signals under 55 experimental conditions. Fitting is performed via an Integer Linear Program (ILP) formulation and solution by standard ILP solvers; a procedure that drastically outperforms previous fitting schemes. Then, knowing the cell's topology, we monitor the same key phosphoprotein signals under the presence of drug and we re-optimize the specific map to reveal drug-induced topology alterations. To prove our case, we make a topology for the hepatocytic cell-line HepG2 and we evaluate the effects of 4 drugs: 3 selective inhibitors for the Epidermal Growth Factor Receptor (EGFR) and a non-selective drug. We confirm effects easily predictable from the drugs' main target (i.e., EGFR inhibitors blocks the EGFR pathway) but we also uncover unanticipated effects due to either drug promiscuity or the cell's specific topology. An interesting finding is that the selective EGFR inhibitor Gefitinib inhibits signaling downstream the Interleukin-1alpha (IL1alpha) pathway; an effect that cannot be extracted from binding affinity-based approaches. Our method represents an unbiased approach to identify drug effects on small to medium size pathways which is scalable to larger topologies with any type of signaling interventions (small molecules, RNAi, etc). The method can reveal drug effects on

  17. Phosphoproteomics reveals the effect of ethylene in soybean root under flooding stress.

    PubMed

    Yin, Xiaojian; Sakata, Katsumi; Komatsu, Setsuko

    2014-12-01

    Flooding has severe negative effects on soybean growth. To explore the flooding-responsive mechanisms in early-stage soybean, a phosphoproteomic approach was used. Two-day-old soybean plants were treated without or with flooding for 3, 6, 12, and 24 h, and root tip proteins were then extracted and analyzed at each time point. After 3 h of flooding exposure, the fresh weight of soybeans increased, whereas the ATP content of soybean root tips decreased. Using a gel-free proteomic technique, a total of 114 phosphoproteins were identified in the root tip samples, and 34 of the phosphoproteins were significantly changed with respect to phosphorylation status after 3 h of flooding stress. Among these phosphoproteins, eukaryotic translation initiation factors were dephosphorylated, whereas several protein synthesis-related proteins were phosphorylated. The mRNA expression levels of sucrose phosphate synthase 1F and eukaryotic translation initiation factor 4 G were down-regulated, whereas UDP-glucose 6-dehydrogenase mRNA expression was up-regulated during growth but down-regulated under flooding stress. Furthermore, bioinformatic protein interaction analysis of flooding-responsive proteins based on temporal phosphorylation patterns indicated that eukaryotic translation initiation factor 4 G was located in the center of the network during flooding. Soybean eukaryotic translation initiation factor 4 G has homology to programmed cell death 4 protein and is implicated in ethylene signaling. The weight of soybeans was increased with treatment by an ethylene-releasing agent under flooding condition, but it was decreased when plants were exposed to an ethylene receptor antagonist. These results suggest that the ethylene signaling pathway plays an important role, via the protein phosphorylation, in mechanisms of plant tolerance to the initial stages of flooding stress in soybean root tips.

  18. Comparative phosphoproteomics reveals components of host cell invasion and post-transcriptional regulation during Francisella infection

    SciTech Connect

    Nakayasu, Ernesto S.; Tempel, Rebecca; Cambronne, Xiaolu A.; Petyuk, Vladislav A.; Jones, Marcus B.; Gritsenko, Marina A.; Monroe, Matthew E.; Yang, Feng; Smith, Richard D.; Adkins, Joshua N.; Heffron, Fred

    2013-09-22

    Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune system that normally leads to cytokine expression and cell death. In previous work, we identified new bacterial factors that were hyper-cytotoxic to macrophages. Four of the identified hyper-cytotoxic strains (lpcC, manB, manC and kdtA) had an impaired lipopolysaccharide (LPS) synthesis and produced an exposed lipid A lacking the O-antigen. These mutants were not only hyper-cytotoxic but also were phagocytosed at much higher rates compared to the wild type parent strain. To elucidate the cellular signaling underlying this enhanced phagocytosis and cell death, we performed a large-scale comparative phosphoproteomic analysis of cells infected with wild-type and delta-lpcC F. novicida. Our data suggest that not only actin but also intermediate filaments and microtubules are important for F. novicida entry into the host cells. In addition, we observed differential phosphorylation of tristetraprolin (TTP), a key component of the mRNA-degrading machinery that controls the expression of a variety of genes including many cytokines. Infection with the delta-lpcC mutant induced the hyper-phosphorylation and inhibition of TTP, leading to the production of cytokines such as IL-1beta and TNF-alpha which may kill the host cells by triggering apoptosis. Together, our data provide new insights for Francisella invasion and a post-transcriptional mechanism that prevents the expression of host immune response factors that controls infection by this pathogen.

  19. Surgical Dissection of the Anterolateral Ligament.

    PubMed

    Daggett, Matthew; Busch, Kyle; Sonnery-Cottet, Bertrand

    2016-02-01

    Recent investigations into the structure and function of the anterolateral ligament (ALL) have resulted in renewed interest in the role of the lateral extra-articular structures in rotational control of the knee. With increased focus on the ALL, debate about the anatomic characteristics, the functional role in knee stability, and even the existence of this lateral structure has ensued. This article describes our dissection method for the ALL. Through careful dissection and precise elevation of the iliotibial band, the ALL can be clearly identified as a distinct structure with an attachment near the lateral epicondyle on the femur and an insertion in a fan-like fashion onto the tibia, between the Gerdy tubercle and the fibular head. This investigation provides the surgeon with anatomic landmarks to use during surgical reconstruction of the ALL. PMID:27274451

  20. Surgical techniques in type A dissection.

    PubMed

    Hussain, Syed T; Svensson, Lars G

    2016-05-01

    Acute aortic dissection is a surgical emergency that must be urgently managed, with the primary goal of restoring flow to the dominant true lumen in the downstream aorta. Our preference at the Cleveland Clinic is for an open distal anastomosis technique without aortic clamping, as it permits more accurate approximation of dissected layers and more homeostatically secure anastomosis. During this procedure we employ right axillary end-to-side graft perfusion, followed by deep hypothermic circulatory arrest and antegrade brain perfusion. The distal anastomosis is performed without felt strips or glue. Critical to achieving a successful outcome is meticulous de-airing of the arch, diligent myocardial protection, and a water-tight anastomosis prior to discontinuing cardiopulmonary bypass. PMID:27386412

  1. Surgical techniques in type A dissection

    PubMed Central

    Hussain, Syed T.

    2016-01-01

    Acute aortic dissection is a surgical emergency that must be urgently managed, with the primary goal of restoring flow to the dominant true lumen in the downstream aorta. Our preference at the Cleveland Clinic is for an open distal anastomosis technique without aortic clamping, as it permits more accurate approximation of dissected layers and more homeostatically secure anastomosis. During this procedure we employ right axillary end-to-side graft perfusion, followed by deep hypothermic circulatory arrest and antegrade brain perfusion. The distal anastomosis is performed without felt strips or glue. Critical to achieving a successful outcome is meticulous de-airing of the arch, diligent myocardial protection, and a water-tight anastomosis prior to discontinuing cardiopulmonary bypass. PMID:27386412

  2. Preparation of Polypropylene Spin Tips Filled with Immobilized Titanium(IV) Ion Monolithic Adsorbent for Robust Phosphoproteome Analysis.

    PubMed

    Liu, Fangjie; Wan, Hao; Liu, Zhongshan; Wang, Hongwei; Mao, Jiawei; Ye, Mingliang; Zou, Hanfa

    2016-05-17

    In this study, we developed a Ti(IV) monolithic spin tip for phosphoproteome analysis of a minute amount of biological sample for the first time. The surface of polypropylene pipet tip was activated by the photoinitiator benzophenone under UV light radiation followed by polymerization of ethylene glycol methacrylate phosphate and bis-acrylamide in the tip to form a porous monolith with reactive phosphate groups. The as-prepared tips grafted with monolithic adsorbent were then chelated with titanium(IV) ion for phosphopeptide enrichment. It was found that the tips enabled fast and efficient capture of phosphopeptides from microscale complex samples. The monolithic tip was demonstrated to have a detection limit as low as 5 fmol β-casein tryptic digest, along with an exceptionally high specificity to capture phosphopeptides from complex tryptic digest mixed with an unphosphorylated protein and a phosphorylated protein at a molar ratio up to 1000:1. When the tip was applied to enrich phosphopeptides from 5 μg of tryptic digest of complex HeLa cell proteins, 1185 high confidence of phosphorylated sites were successfully identified with the specificity as high as 92.5%. So far, this is the most sensitive phosphoproteomics analysis using a standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) system for proteome-wide phosphorylation analysis in mammalian cells. PMID:27101427

  3. Biosynthesis and Regulation of Wheat Amylose and Amylopectin from Proteomic and Phosphoproteomic Characterization of Granule-binding Proteins

    PubMed Central

    Chen, Guan-Xing; Zhou, Jian-Wen; Liu, Yan-Lin; Lu, Xiao-Bing; Han, Cai-Xia; Zhang, Wen-Ying; Xu, Yan-Hao; Yan, Yue-Ming

    2016-01-01

    Waxy starch has an important influence on the qualities of breads. Generally, grain weight and yield in waxy wheat (Triticum aestivum L.) are significantly lower than in bread wheat. In this study, we performed the first proteomic and phosphoproteomic analyses of starch granule-binding proteins by comparing the waxy wheat cultivar Shannong 119 and the bread wheat cultivar Nongda 5181. These results indicate that reduced amylose content does not affect amylopectin synthesis, but it causes significant reduction of total starch biosynthesis, grain size, weight and grain yield. Two-dimensional differential in-gel electrophoresis identified 40 differentially expressed protein (DEP) spots in waxy and non-waxy wheats, which belonged mainly to starch synthase (SS) I, SS IIa and granule-bound SS I. Most DEPs involved in amylopectin synthesis showed a similar expression pattern during grain development, suggesting relatively independent amylose and amylopectin synthesis pathways. Phosphoproteome analysis of starch granule-binding proteins, using TiO2 microcolumns and LC-MS/MS, showed that the total number of phosphoproteins and their phosphorylation levels in ND5181 were significantly higher than in SN119, but proteins controlling amylopectin synthesis had similar phosphorylation levels. Our results revealed the lack of amylose did not affect the expression and phosphorylation of the starch granule-binding proteins involved in amylopectin biosynthesis. PMID:27604546

  4. In vivo Phosphoproteome of Human Skeletal Muscle Revealed by Phosphopeptide Enrichment and HPLC-ESI-MS/MS

    PubMed Central

    Højlund, Kurt; Bowen, Benjamin P.; Hwang, Hyonson; Flynn, Charles R.; Madireddy, Lohith; Thangiah, Geetha; Langlais, Paul; Meyer, Christian; Mandarino, Lawrence J.; Yi, Zhengping

    2009-01-01

    Protein phosphorylation plays an essential role in signal transduction pathways that regulate substrate and energy metabolism, contractile function, and muscle mass in human skeletal muscle. Abnormal phosphorylation of signaling enzymes has been identified in insulin resistant muscle using phosphoepitope-specific antibodies, but its role in other skeletal muscle disorders remains largely unknown. This may be in part due to insufficient knowledge of relevant targets. Here, we therefore present the first large-scale in vivo phosphoproteomic study of human skeletal muscle from 3 lean, healthy volunteers. Trypsin digestion of 3-5 mg human skeletal muscle protein was followed by phosphopeptide enrichment using SCX and TiO2. The resulting phosphopeptides were analyzed by HPLC-ESI-MS/MS. Using this unbiased approach, we identified 306 distinct in vivo phosphorylation sites in 127 proteins, including 240 phosphoserines, 53 phosphothreonines and 13 phosphotyrosines in at least 2 out of 3 subjects. In addition, 61 ambiguous phosphorylation sites were identified in at least 2 out of 3 subjects. The majority of phosphoproteins detected are involved in sarcomeric function, excitation-contraction coupling (the Ca2+-cycle), glycolysis and glycogen metabolism. Of particular interest, we identified multiple novel phosphorylation sites on several sarcomeric Z-disc proteins known to be involved in signaling and muscle disorders. These results provide numerous new targets for the investigation of human skeletal muscle phosphoproteins in health and disease and demonstrate feasibility of phosphoproteomics research of human skeletal muscle in vivo. PMID:19764811

  5. Biosynthesis and Regulation of Wheat Amylose and Amylopectin from Proteomic and Phosphoproteomic Characterization of Granule-binding Proteins.

    PubMed

    Chen, Guan-Xing; Zhou, Jian-Wen; Liu, Yan-Lin; Lu, Xiao-Bing; Han, Cai-Xia; Zhang, Wen-Ying; Xu, Yan-Hao; Yan, Yue-Ming

    2016-01-01

    Waxy starch has an important influence on the qualities of breads. Generally, grain weight and yield in waxy wheat (Triticum aestivum L.) are significantly lower than in bread wheat. In this study, we performed the first proteomic and phosphoproteomic analyses of starch granule-binding proteins by comparing the waxy wheat cultivar Shannong 119 and the bread wheat cultivar Nongda 5181. These results indicate that reduced amylose content does not affect amylopectin synthesis, but it causes significant reduction of total starch biosynthesis, grain size, weight and grain yield. Two-dimensional differential in-gel electrophoresis identified 40 differentially expressed protein (DEP) spots in waxy and non-waxy wheats, which belonged mainly to starch synthase (SS) I, SS IIa and granule-bound SS I. Most DEPs involved in amylopectin synthesis showed a similar expression pattern during grain development, suggesting relatively independent amylose and amylopectin synthesis pathways. Phosphoproteome analysis of starch granule-binding proteins, using TiO2 microcolumns and LC-MS/MS, showed that the total number of phosphoproteins and their phosphorylation levels in ND5181 were significantly higher than in SN119, but proteins controlling amylopectin synthesis had similar phosphorylation levels. Our results revealed the lack of amylose did not affect the expression and phosphorylation of the starch granule-binding proteins involved in amylopectin biosynthesis. PMID:27604546

  6. Alterations in the Cerebellar (Phospho)Proteome of a Cyclic Guanosine Monophosphate (cGMP)-dependent Protein Kinase Knockout Mouse*

    PubMed Central

    Corradini, Eleonora; Vallur, Raghavan; Raaijmakers, Linsey M.; Feil, Susanne; Feil, Robert; Heck, Albert J. R.; Scholten, Arjen

    2014-01-01

    The cyclic nucleotide cyclic guanosine monophosphate (cGMP) plays an important role in learning and memory, but its signaling mechanisms in the mammalian brain are not fully understood. Using mass-spectrometry-based proteomics, we evaluated how the cerebellum adapts its (phospho)proteome in a knockout mouse model of cGMP-dependent protein kinase type I (cGKI). Our data reveal that a small subset of proteins in the cerebellum (∼3% of the quantified proteins) became substantially differentially expressed in the absence of cGKI. More changes were observed at the phosphoproteome level, with hundreds of sites being differentially phosphorylated between wild-type and knockout cerebellum. Most of these phosphorylated sites do not represent known cGKI substrates. An integrative computational network analysis of the data indicated that the differentially expressed proteins and proteins harboring differentially phosphorylated sites largely belong to a tight network in the Purkinje cells of the cerebellum involving important cGMP/cAMP signaling nodes (e.g. PDE5 and PKARIIβ) and Ca2+ signaling (e.g. SERCA3). In this way, removal of cGKI could be linked to impaired cerebellar long-term depression at Purkinje cell synapses. In addition, we were able to identify a set of novel putative (phospho)proteins to be considered in this network. Overall, our data improve our understanding of cerebellar cGKI signaling and suggest novel players in cGKI-regulated synaptic plasticity. PMID:24925903

  7. Future directions of duodenal endoscopic submucosal dissection

    PubMed Central

    Matsumoto, Satohiro; Miyatani, Hiroyuki; Yoshida, Yukio

    2015-01-01

    Endoscopic therapies for lesions of the duodenum are technically more difficult than those for lesions of the other parts of the gastrointestinal tract due to the anatomical features of the duodenum, and the incidence rate of complications such as perforation and bleeding is also higher. These aforementioned trends were especially noticeable for the case of duodenal endoscopic submucosal dissection (ESD). The indication for ESD of duodenal tumors should be determined by assessment of the histopathology, macroscopic morphology, and diameter of the tumors. The three types of candidate lesions for endoscopic therapy are adenoma, carcinoma, and neuroendocrine tumors. For applying endoscopic therapies to duodenal lesions, accurate preoperative histopathological diagnosis is necessary. The most important technical issue in duodenal ESD is the submucosal dissection process. In duodenal ESD, a short needle-type knife is suitable for the mucosal incision and submucosal dissection processes, and the Small-caliber-tip Transparent hood is an important tool. After endoscopic therapies, the wound should be closed by clipping in order to prevent complications such as secondary hemorrhage and delayed perforation. At present, the criteria for selection between ESD and EMR vary among institutions. The indications for ESD should be carefully considered. Duodenal ESD should have limitations, such as the need for its being performed by experts with abundant experience in performing the procedure. PMID:25901218

  8. "Ostrich sign" indicates bilateral vertebral artery dissection.

    PubMed

    Rose, David Z; Husain, M Rizwan

    2012-11-01

    Vertebral artery dissections (VADs) comprise about 2% of ischemic strokes and can be associated with trauma, chiropractic manipulation, motor vehicle collisions, whiplash, amusement park rides, golfing, and other motion-induced injuries to the neck. We present a case of bilateral extracranial VAD as a complication of conducting an orchestra. To our knowledge, this has not been documented in the literature. Conceivably, vigorous neck twisting in an inexperienced, amateur conductor may place excessive rotational forces upon mobile portions of the verterbral arteries, tear the intima, deposit subintimal blood that extends longitudinally, and cause neck pain and/or posterior fossa ischemic symptoms. Magnetic resonance angiography examinations of axially oriented slices of bilateral VADs resemble the face of an ostrich. This observation is similar to the "puppy sign," in which bilateral internal carotid artery dissections resemble the face of a dog. Craniocervical dissections of either the carotid or vertebral arteries have the potential to form an aneurysm, cause artery-to-artery embolism, or completely occlude the parent artery, resulting in an ischemic stroke. Because bilateral VADs in axial magnetic resonance angiographic sections stand out like the eyes of an ostrich, and because the fast identification of VADs is so critical, we eponymize this image the "ostrich sign."

  9. Building mental models by dissecting physical models.

    PubMed

    Srivastava, Anveshna

    2016-01-01

    When students build physical models from prefabricated components to learn about model systems, there is an implicit trade-off between the physical degrees of freedom in building the model and the intensity of instructor supervision needed. Models that are too flexible, permitting multiple possible constructions require greater supervision to ensure focused learning; models that are too constrained require less supervision, but can be constructed mechanically, with little to no conceptual engagement. We propose "model-dissection" as an alternative to "model-building," whereby instructors could make efficient use of supervisory resources, while simultaneously promoting focused learning. We report empirical results from a study conducted with biology undergraduate students, where we demonstrate that asking them to "dissect" out specific conceptual structures from an already built 3D physical model leads to a significant improvement in performance than asking them to build the 3D model from simpler components. Using questionnaires to measure understanding both before and after model-based interventions for two cohorts of students, we find that both the "builders" and the "dissectors" improve in the post-test, but it is the latter group who show statistically significant improvement. These results, in addition to the intrinsic time-efficiency of "model dissection," suggest that it could be a valuable pedagogical tool. PMID:26712513

  10. Advanced endoscopic submucosal dissection with traction

    PubMed Central

    Imaeda, Hiroyuki; Hosoe, Naoki; Kashiwagi, Kazuhiro; Ohmori, Tai; Yahagi, Naohisa; Kanai, Takanori; Ogata, Haruhiko

    2014-01-01

    Endoscopic submucosal dissection (ESD) has been established as a standard treatment for early stage gastric cancer (EGC) in Japan and has spread worldwide. ESD has been used not only for EGC but also for early esophageal and colonic cancers. However, ESD is associated with several adverse events, such as bleeding and perforation, which requires more skill. Adequate tissue tension and clear visibility of the tissue to be dissected are important for effective and safe dissection. Many ESD methods using traction have been developed, such as clip-with-line method, percutaneous traction method, sinker-assisted method, magnetic anchor method, external forceps method, internal-traction method, double-channel-scope method, outerroute method, double-scope method, endoscopic-surgical-platform, and robot-assisted method. Each method has both advantages and disadvantages. Robotic endoscopy, enabling ESD with a traction method, will become more common due to advances in technology. In the near future, simple, noninvasive, and effective ESD using traction is expected to be developed and become established as a worldwide standard treatment for superficial gastrointestinal neoplasias. PMID:25031787

  11. Dissection of aorta: a pediatric case report.

    PubMed

    Serdaroğlu, Gül; Levent, Ertürk; Yurtsever, Serap; Calkavur, Tanzer; Yünten, Nilgün; Aydoğdu, Sema

    2002-01-01

    We present a 15-year-old boy who developed sudden walking disability and sensory loss. He could not stand up on his feet and had no feeling following a sudden fall while playing basketball. He had been referred to a local hospital with these symptoms. In his physical examination absence of deep tendon reflexes and sensory loss were noted. His arterial blood pressure was 210/160 mmHg. He was transferred to our hospital with these findings and diagnosis of Guillain-Barré syndrome and hypertensive encephalopathy. There was sudden onset of sensory loss, walking disability and history of trauma. In the following hours hematuria, back pain and lower extremity ischemia developed. We suspected spinal artery injury based on the findings. Dissection of descending aorta was established with the help of magnetic resonance imaging of spinal region and contrasted aortography. The patient went to surgery immediately. He was lost on the second day after operation because of malperfusion. We report this case because dissecting aorta is very rare in the pediatric age group. High index of suspicion and early aortography are needed to diagnose aorta dissection.

  12. Simultaneous onset of anterior and middle cerebral artery dissections with an old vertebral artery dissection.

    PubMed

    Kato, Tatsuya; Yagi, Takashi; Yoshioka, Hideyuki; Ogiwara, Masakazu; Horikoshi, Toru; Kinouchi, Hiroyuki

    2013-11-01

    Multiple arterial dissections in the anterior circulation with simultaneous onset are extremely rare. We report a patient with infarctions caused by simultaneous arterial dissections in the right anterior cerebral artery and the left middle cerebral artery and discuss the characteristic feature of this vascular disorder. A 53-year-old woman presented with a severe headache and a mild aphasia. Magnetic resonance imaging revealed multiple acute cerebral infarctions in the left temporal and right frontal lobes. The initial angiographic findings revealed arterial dissections of the anterior cerebral, left middle cerebral, and right vertebral arteries. The follow-up angiographic examination found improvement of the stenosis in both the anterior cerebral and middle cerebral arteries. We have concluded that the lesion of the vertebral artery was not in an acute stage, because no interval change was seen during the radiologic evaluation. She underwent conservative therapy, and her symptoms disappeared. Multiple arterial dissections are rare, especially those developing simultaneously in different arteries. This is the first case of multiple arterial dissections of the different arteries in the anterior circulation manifesting cerebral infarction simultaneously.

  13. Extracranial and intracranial vertebrobasilar dissections: diagnosis and prognosis

    PubMed Central

    de Bray, J M; Penisson-Besnier, I; Dubas, F; Emile, J

    1997-01-01

    OBJECTIVES—To compare the diagnosis and prognosis of extracranial versus intracranial vertebral artery dissections without intracerebral haemorrhage.
METHODS—Twenty two vertebral artery dissections were defined by intra-arterial angiography and classified in two groups: group 1,nine extracranial dissections (seven patients) and group 2, 13intracranial dissections (nine patients), involving the basilar artery in five cases. Bilateral dissections were found in 38% of the population. Before angiography, all the patients had been investigated by continuous wave Doppler, colour coded Doppler, and transcranial Doppler. Mean follow up was 44months.
RESULTS—The two most important symptoms of both dissections (81% of patients) were unbearable pain preceding stroke and progressive onset of stroke within a few hours. Severe ultrasonic abnormalities were present in 94% of the patients whereas specific ultrasonic signs (segmental dilation with eccentric channel) were rare (19%) in both groups. Major strokes and brainstem strokes represented respectively 67% and 78% in intracranial versus 43% and 29% in extracranial dissections. Severe sequelae (permanent disabling motor or cerebellar deficit) were more often associated with intracranial (44%) than with extracranial dissections (14%). No recurrence of dissection and no cerebral haemorrhage were found under heparin. Significant factors of poor outcome (P< 0.05) were the initial severity of the stroke and the bilateral location of dissections.
CONCLUSION—The combination of a pain and a progressive onset of the stroke, corroborated by ultrasonic findings, could have helped to recognise most of these types of dissections. Intracranial dissections have a poorer prognosis than extracranial dissections.

 PMID:9221967

  14. Background music in the dissection laboratory: impact on stress associated with the dissection experience.

    PubMed

    Anyanwu, Emeka G

    2015-06-01

    Notable challenges, such as mental distress, boredom, negative moods, and attitudes, have been associated with learning in the cadaver dissection laboratory (CDL). The ability of background music (BM) to enhance the cognitive abilities of students is well documented. The present study was designed to investigate the impact of BM in the CDL and on stress associated with the dissection experience. After 8 wk of normal dissection without BM, various genres of BM were introduced into the cadaver dissection sessions of 260 medical and dental students for 3 wk. Feedback on the impact of BM on students in the CDL and students' attitude were accessed using a questionnaire. Psychological stress assessment was done using Psychological Stress Measure 9. Two batches of 30 students each were made to dissect same areas of the body for 2 h, one batch with BM playing and the other batch without. The same examination was given to both groups at the end. Over 90% of the participants expressed a desire to incorporate BM into the CDL; 87% of the sampled population that expressed love for music also reported BM to be a very useful tool that could be used to enhance learning conditions in the CDL. A strong positive relationship was established between love for music and its perception as a tool for learning in the CDL (P < 0.001). Students that studied under the influence of BM had significantly higher scores (P < 0.001) in the overall examination result. BM reduced the level of stress associated with the dissection experience by ∼33%. PMID:26031725

  15. Background music in the dissection laboratory: impact on stress associated with the dissection experience.

    PubMed

    Anyanwu, Emeka G

    2015-06-01

    Notable challenges, such as mental distress, boredom, negative moods, and attitudes, have been associated with learning in the cadaver dissection laboratory (CDL). The ability of background music (BM) to enhance the cognitive abilities of students is well documented. The present study was designed to investigate the impact of BM in the CDL and on stress associated with the dissection experience. After 8 wk of normal dissection without BM, various genres of BM were introduced into the cadaver dissection sessions of 260 medical and dental students for 3 wk. Feedback on the impact of BM on students in the CDL and students' attitude were accessed using a questionnaire. Psychological stress assessment was done using Psychological Stress Measure 9. Two batches of 30 students each were made to dissect same areas of the body for 2 h, one batch with BM playing and the other batch without. The same examination was given to both groups at the end. Over 90% of the participants expressed a desire to incorporate BM into the CDL; 87% of the sampled population that expressed love for music also reported BM to be a very useful tool that could be used to enhance learning conditions in the CDL. A strong positive relationship was established between love for music and its perception as a tool for learning in the CDL (P < 0.001). Students that studied under the influence of BM had significantly higher scores (P < 0.001) in the overall examination result. BM reduced the level of stress associated with the dissection experience by ∼33%.

  16. Dissecting the Dissection: Towards More Comprehensive Decision-Making Methodology for Thoracic Aortic Disease.

    PubMed

    Sherif, Hisham M F

    2015-06-01

    Aortic dissection remains one of the most devastating diseases. Current practice guidelines provide diagnostic and therapeutic interventions based primarily on the aortic diameter. The level of evidence supporting these recommendations is Level C or "Expert Opinion" Since aortic dissection is a catastrophic structural failure, its investigation along the guidelines of accident investigation may offer a useful alternative, utilizing process mapping and root-cause analysis methodology. Since the objective of practice guidelines is to address the risk of serious events, on the utilization of a probabilistic predictive modeling methodology, using bioinformatics tools, may offer a more comprehensive risk assessment. PMID:27069940

  17. The many faces of aortic dissections: Beware of unusual presentations.

    PubMed

    Scaglione, Mariano; Salvolini, Luca; Casciani, Emanuele; Giovagnoni, Andrea; Mazzei, Maria Antonietta; Volterrani, Luca

    2008-03-01

    Aortic dissection is gaining recognition in Western societies, and it is being diagnosed with increasing frequency. New diagnostic imaging modalities, longer life expectancy, as well as the increase in the number patients suffering from hypertension have all contributed to the growing awareness of aortic dissection. Nevertheless, as many as 30% of patients ultimately diagnosed with acute dissection are first thought to be suffering from something else. The increased availability and use of multidetector computed tomography has led to the incidental discovery of aortic dissection in very different settings. This article focuses on unusual presentations of painless aortic dissection. It is important for radiologists to remember that aortic dissections may present in different ways, not only as an acute, critical fatality but also with subtle, unusual signs and symptoms, which apparently do not seem to be strictly related to aortic diseases. PMID:17950552

  18. Intimal Flap Vegetation Following Aortic Root Re-dissection

    PubMed Central

    Osada, Hiroaki; Nakajima, Hiroyuki; Meshii, Katsuaki; Ohnaka, Motoaki

    2016-01-01

    A 75-year-old man who had undergone ascending aorta replacement for acute Type A aortic dissection presented with a recurring high fever. Transesophageal echocardiography revealed that a vegetation had formed on the re-dissected intimal flap of the noncoronary sinus of Valsalva. This didactic case suggests that antibiotic prophylactic measures be considered for aortic dissection flaps as for irregular valves susceptible to infective endocarditis.

  19. Rare Seagull Cooing Murmur from Acute Aortic Dissection

    PubMed Central

    Zhao, J; Cheng, Z; Quan, X; Zhao, Z

    2015-01-01

    ABSTRACT Acute aortic dissection is a rare but potentially fatal disease. The early recognition of this disease is important for timely treatment. Some signs and symptoms, such as past history of hypertension, tearing pain and pulselessness, can provide valuable clues to the diagnosis of this disease. In this case study, the mechanism of a seagull murmur from aortic dissection is first described. This information is potentially useful for the differential diagnosis of dissection. PMID:26426186

  20. Dissecting calcium oscillators in plant cells.

    PubMed

    Harper, J F

    2001-09-01

    To understand Ca2+ signaling, we need to identify all the Ca2+ transporters and their regulatory components. The first Ca2+ transporters to be cloned from plants and shown to have regulated activity were calmodulin-dependent Ca2+ -pumps. The regulation of these pumps suggests that being able to change the rate of Ca2+ efflux is important for Ca2+ signaling. The identification of pumps and antiporters in different subcellular locations is helping to dissect the complexities of Ca2+ signaling in plants.

  1. Aortic dissection presenting as gait disturbance: a case report.

    PubMed

    Estreicher, Michael; Portale, Joseph; Lopez, Bernard

    2013-01-01

    Emergency medicine dogma traditionally teaches that aortic dissection presents as tearing chest pain, radiating to the back. This case report describes a 55-year-old woman presenting with a left homonymous hemianopsia and resultant gait disturbance. Initial head computed tomography demonstrated a right parietal infarct, and chest radiograph demonstrated a markedly widened mediastinum. Acute Stanford type A aortic dissection was subsequently confirmed. This report provides further evidence for atypical, painless presentations of aortic dissection. Given recent literature on the increasing prevalence of painless dissection, the disease entity should be included in the differential diagnosis for stroke, and a simple portable chest x-ray should always be obtained before administering thrombolytics.

  2. Dissection videos do not improve anatomy examination scores.

    PubMed

    Mahmud, Waqas; Hyder, Omar; Butt, Jamaal; Aftab, Arsalan

    2011-01-01

    In this quasi-experimental study, we describe the effect of showing dissection videos on first-year medical students' performance in terms of test scores during a gross anatomy course. We also surveyed students' perception regarding the showing of dissection videos. Two hundred eighty-seven first-year medical students at Rawalpindi Medical College in Pakistan, divided into two groups, dissected one limb in first term and switched over to the other limb in the second term. During the second term, instruction was supplemented by dissection videos. Second-term anatomy examination marks were compared with first-term scores and with results from first-year medical students in previous years. Multiple linear regression analysis was performed, with term scores (continuous, 0-200) as the dependent variable. Students shown dissection videos scored 1.26 marks higher than those not shown. The relationship was not statistically significant (95% CI: -1.11, 3.70; P = 0.314). Ninety-three percent of students favored regular inclusion of dissection videos in curriculum, and 50% termed it the best source for learning gross anatomy. Seventy-six percent of students did not perform regular cadaver dissection. The most frequent reason cited for not performing regular dissection was high student-cadaver ratio. Dissection videos did not improve performance on final examination scores; however, students favored their use.

  3. Acute aortic dissection diagnosed after embalming: macroscopic and microscopic findings.

    PubMed

    Savall, Frédéric; Dedouit, Fabrice; Piercecchi-Marti, Marie-Dominique; Leonetti, Georges; Rougé, Daniel; Telmon, Norbert

    2014-09-01

    A 58-year-old man died suddenly in Madagascar and poisoning was suspected. The body was embalmed after death and the general state of preservation was good. We found a major aortic dissection with a large false lumen from the aortic root to the common iliac arteries and a hemopericardium with formalinized blood clot. The intimal tear was on the ascending aorta, and an intramural hemorrhage was noted at the right coronary artery, attesting to a retrograde dissection. Microscopic studies confirmed aortic dissection with extensive intramural hemorrhage and also confirmed the retrograde dissection to the right coronary artery with a reduction of 90% of the true lumen. Classically, aortic dissection occurs in individuals with hypertension and individuals with genetic disorders of collagen formation. The diagnosis is often first established at the postmortem examination. Aortic dissection is therefore dealt with largely in necropsy studies. The usual cause of death is rupture into the pericardial sac. One case of bloodless dissection has been reported but the sudden death was explained by acute myocardial ischemia secondary to dissection of the left coronary artery. In our case, we found major hemopericardium and also intramural hemorrhage at the right coronary artery. We were able to make the diagnosis of aortic dissection and exclude the suspicion of homicide 15 days after death and after embalming.

  4. Acute aortic dissection diagnosed after embalming: macroscopic and microscopic findings.

    PubMed

    Savall, Frédéric; Dedouit, Fabrice; Piercecchi-Marti, Marie-Dominique; Leonetti, Georges; Rougé, Daniel; Telmon, Norbert

    2014-09-01

    A 58-year-old man died suddenly in Madagascar and poisoning was suspected. The body was embalmed after death and the general state of preservation was good. We found a major aortic dissection with a large false lumen from the aortic root to the common iliac arteries and a hemopericardium with formalinized blood clot. The intimal tear was on the ascending aorta, and an intramural hemorrhage was noted at the right coronary artery, attesting to a retrograde dissection. Microscopic studies confirmed aortic dissection with extensive intramural hemorrhage and also confirmed the retrograde dissection to the right coronary artery with a reduction of 90% of the true lumen. Classically, aortic dissection occurs in individuals with hypertension and individuals with genetic disorders of collagen formation. The diagnosis is often first established at the postmortem examination. Aortic dissection is therefore dealt with largely in necropsy studies. The usual cause of death is rupture into the pericardial sac. One case of bloodless dissection has been reported but the sudden death was explained by acute myocardial ischemia secondary to dissection of the left coronary artery. In our case, we found major hemopericardium and also intramural hemorrhage at the right coronary artery. We were able to make the diagnosis of aortic dissection and exclude the suspicion of homicide 15 days after death and after embalming. PMID:24684562

  5. Robotic radical prostatectomy: advantages of an initial posterior dissection.

    PubMed

    Zorn, Kevin C

    2008-09-01

    During robotic radical prostatectomy (RRP), many surgeons currently employ the modified-Montsouris technique as initially described by Menon in 2002 with initial anterior prostate dissection. The anterior approach simulates the routine retropubic technique which open surgeons feel most comfortable with. Unfortunately, we observed early on in our experience that dissection of the seminal vesicles (SV) and vas deferens (VD) through a limited sized bladder neck posed limitations on working space and anatomic differentiation. As such, we have continued using a posterior-first dissection for several specific advantages. Herein, we describe our initial posterior dissection during RRP and discuss potential advantages of this approach, particularly for novice robotic surgeons. PMID:27628248

  6. Computed Tomography Imaging Artifact Simulating Type A Aortic Dissection

    PubMed Central

    Chou, Alan S.; Ziganshin, Bulat A.; Elefteriades, John A.

    2016-01-01

    Contrast-enhanced computed tomography (CT) is an effective tool for assessment of thoracic aortic disease in the modern era. Here, we describe a case of Type A aortic dissection incidentally detected by CT in a 63-year old man. Upon more precise imaging with electrocardiography (ECG)-gated CT, the dissection vanished, revealing it to be an aortic motion artifact. This report highlights the importance of motion artifacts mimicking a dissection flap. CT imaging gated with ECG can distinguish a dissection flap from an artifact.

  7. Doing Dissections Differently: A Structured, Peer-Assisted Learning Approach to Maximizing Learning in Dissections

    ERIC Educational Resources Information Center

    Hall, Emma R.; Davis, Rachel C.; Weller, Renate; Powney, Sonya; Williams, Sarah B.

    2013-01-01

    Areas of difficulty faced by our veterinary medicine students, with respect to their learning in dissection classes, were identified. These challenges were both general adult-learning related and specific to the discipline of anatomy. Our aim was to design, implement, and evaluate a modified reciprocal peer-assisted/team-based learning…

  8. Background Music in the Dissection Laboratory: Impact on Stress Associated with the Dissection Experience

    ERIC Educational Resources Information Center

    Anyanwu, Emeka G.

    2015-01-01

    Notable challenges, such as mental distress, boredom, negative moods, and attitudes, have been associated with learning in the cadaver dissection laboratory (CDL). The ability of background music (BM) to enhance the cognitive abilities of students is well documented. The present study was designed to investigate the impact of BM in the CDL and on…

  9. The Effect of a Prior Dissection Simulation on Middle School Students' Dissection Performance and Understanding of the Anatomy and Morphology of the Frog

    NASA Astrophysics Data System (ADS)

    Akpan, Joseph Paul; Andre, Thomas

    1999-06-01

    Science teachers, school administrators, educators, and the scientific community are faced with ethical controversies over animal dissection in classrooms. Simulation has been proposed as a way of dealing with this issue. One intriguing previous finding was that use of an interactive videodisc dissection facilitated performance on a subsequent actual dissection. This study examined the prior use of simulation of frog dissection in improving students' actual dissection performance and learning of frog anatomy and morphology. There were three experimental conditions: simulation before dissection (SBD); dissection before simulation (DBS); or dissection-only (DO). Results of the study indicated that students receiving SBD performed significantly better than students receiving DBS or DO on both actual dissection and knowledge of the anatomy and morphology. Students' attitudes toward the use of animals for dissection did not change significantly from pretest to posttest and did not interact with treatment. The genders did not differ in achievement, but males were more favorable towards dissection and computers than were females.

  10. Molecular Mechanisms of Thoracic Aortic Dissection

    PubMed Central

    Wu, Darrell; Shen, Ying H.; Russell, Ludivine; Coselli, Joseph S.; LeMaire, Scott A.

    2013-01-01

    Thoracic aortic dissection (TAD) is a highly lethal vascular disease. In many patients with TAD, the aorta progressively dilates and ultimately ruptures. Dissection formation, progression, and rupture cannot be reliably prevented pharmacologically because the molecular mechanisms of aortic wall degeneration are poorly understood. The key histopathologic feature of TAD is medial degeneration, a process characterized by smooth muscle cell depletion and extracellular matrix degradation. These structural changes have a profound impact on the functional properties of the aortic wall and can result from excessive protease-mediated destruction of the extracellular matrix, altered signaling pathways, and altered gene expression. Review of the literature reveals differences in the processes that lead to ascending versus descending and sporadic versus hereditary TAD. These differences add to the complexity of this disease. Although tremendous progress has been made in diagnosing and treating TAD, a better understanding of the molecular, cellular, and genetic mechanisms that cause this disease is necessary to developing more effective preventative and therapeutic treatment strategies. PMID:23856125

  11. Molecular dissection of developmental behavior of plant height in rice (Oryza sativa L.).

    PubMed Central

    Yan, J; Zhu, J; He, C; Benmoussa, M; Wu, P

    1998-01-01

    A doubled haploid population of 123 lines from IR64/Azucena was used to dissect the developmental behavior and genotype by environment interaction for plant height by conditional and unconditional quantitative trait loci (QTL) mapping methods in rice. It was shown that the number of QTL detected was different at various measuring stages. Some QTL could be detected at all stages and some only at one or several stages. More QTL could be found on the basis of time-dependent measures of different stages. By conditional QTL mapping of time-dependent measures, it is possible to reveal dynamic gene expression for quantitative traits. Mapping QTL for genetic main effects and GE interaction effects could help us in understanding the nature of QTL x environment interaction for the development of quantitative traits. PMID:9799277

  12. Genetic dissection of agronomic traits in Capsicum baccatum var. pendulum.

    PubMed

    Moulin, M M; Rodrigues, R; Bento, C S; Gonçalves, L S A; Santos, J O; Sudré, C P; Viana, A P

    2015-03-20

    Genetic mapping is very useful for dissecting complex agronomic traits. Genetic mapping allows for identification of quantitative trait loci (QTL), provide knowledge on a gene position and its adjacent region, and enable prediction of evolutionary mechanisms, in addition to contributing to synteny studies. The aim of this study was to predict genetic values associated with different agronomic traits evaluated in an F2 population of Capsicum baccatum var. pendulum. Previously, a reference genetic map for C. baccatum was constructed, which included 183 markers (42 microsatellite, 85 inter-simple sequence repeat, and 56 random amplification of polymorphic DNA) arranged in 16 linkage groups. The map was used to identify QTL associated with 11 agronomic traits, including plant height, crown diameter, number of days to flowering, days to fruiting, number of fruits per plant, average fruit weight, fruit length, fruit diameter, fruit pulp thickness, soluble solids, and fruit dry weight. QTL mapping was performed by standard interval mapping. The number of small QTL effects ranged from 3-11, with a total of 61 QTL detected in 9 linkage groups. This is the first report involving QTL analysis for C. baccatum species.

  13. Dissection of miRNA pathways using arabidopsis mesophyll protoplasts.

    PubMed

    Martinho, Cláudia; Confraria, Ana; Elias, Carlos Alexandre; Crozet, Pierre; Rubio-Somoza, Ignacio; Weigel, Detlef; Baena-González, Elena

    2015-02-01

    MicroRNAs (miRNAs) control gene expression mostly post-transcriptionally by guiding transcript cleavage and/or translational repression of complementary mRNA targets, thereby regulating developmental processes and stress responses. Despite the remarkable expansion of the field, the mechanisms underlying miRNA activity are not fully understood. In this article, we describe a transient expression system in Arabidopsis mesophyll protoplasts, which is highly amenable for the dissection of miRNA pathways. We show that by transiently overexpressing primary miRNAs and target mimics, we can manipulate miRNA levels and consequently impact on their targets. Furthermore, we developed a set of luciferase-based sensors for quantifying miRNA activity that respond specifically to both endogenous and overexpressed miRNAs and target mimics. We demonstrate that these miRNA sensors can be used to test the impact of putative components of the miRNA pathway on miRNA activity, as well as the impact of specific mutations, by either overexpression or the use of protoplasts from the corresponding mutants. We further show that our miRNA sensors can be used for investigating the effect of chemicals on miRNA activity. Our cell-based transient expression system is fast and easy to set up, and generates quantitative results, being a powerful tool for assaying miRNA activity in vivo.

  14. Diagnosis of Aortic Dissection in Emergency Department Patients is Rare

    PubMed Central

    Alter, Scott M.; Eskin, Barnet; Allegra, John R.

    2015-01-01

    Introduction Aortic dissection is a rare event. While the most frequent symptom is chest pain, that is a common emergency department (ED) chief complaint and other diseases causing chest pain occur much more often. Furthermore, 20% of dissections are without chest pain and 6% are painless. For these reasons, diagnosing dissections may be challenging. Our goal was to determine the number of total ED and atraumatic chest pain patients for every aortic dissection diagnosed by emergency physicians. Methods Design: Retrospective cohort. Setting: 33 suburban and urban New York and New Jersey EDs with annual visits between 8,000 and 80,000. Participants: Consecutive patients seen by emergency physicians from 1-1-1996 through 12-31-2010. Observations: We identified aortic dissection and atraumatic chest pain patients using the International Classification of Diseases 9th Revision and Clinical Modification codes. We then calculated the number of total ED and atraumatic chest pain patients for every aortic dissection, along with 95% confidence intervals (CIs). Results From a database of 9.5 million ED visits, we identified 782 aortic dissections or one for every 12,200 (95% CI [11,400–13,100]) visits. The mean age of dissection patients was 66±16 years and 38% were female. There were 763,000 (8%) with atraumatic chest pain diagnoses. Thus, there is one dissection for every 980 (95% CI [910–1,050]) atraumatic chest pain patients. Conclusion The diagnosis of aortic dissections by emergency physicians is rare and challenging. An emergency physician seeing 3,000 to 4,000 patients a year would diagnose an aortic dissection approximately every three to four years. PMID:26587083

  15. Quantitative analysis

    PubMed Central

    Nevin, John A.

    1984-01-01

    Quantitative analysis permits the isolation of invariant relations in the study of behavior. The parameters of these relations can serve as higher-order dependent variables in more extensive analyses. These points are illustrated by reference to quantitative descriptions of performance maintained by concurrent schedules, multiple schedules, and signal-detection procedures. Such quantitative descriptions of empirical data may be derived from mathematical theories, which in turn can lead to novel empirical analyses so long as their terms refer to behavioral and environmental events. Thus, quantitative analysis is an integral aspect of the experimental analysis of behavior. PMID:16812400

  16. The Effect of a Prior Dissection Simulation on Middle School Students' Dissection Performance and Understanding of the Anatomy and Morphology of the Frog.

    ERIC Educational Resources Information Center

    Akpan, Joseph Paul; Andre, Thomas

    1999-01-01

    Reports on a study that examined the prior use of simulation on frog dissection in improving students' actual dissection performance and learning of frog anatomy and morphology. Finds that students who performed the simulation before dissection performed significantly better than students who performed dissection before using the simulation or…

  17. Primary Percutaneous Coronary Intervention in Chronic Type A Aortic Dissection

    PubMed Central

    Farag, Ahmed; Chauhan, Aneesha; Avinash, Sunita; Liew, Chee K; Eichhöfer, Jonas; Chauhan, Anoop

    2016-01-01

    This case illustrates the complexities of performing a primary percutaneous intervention in a patient with chronic Type A aortic aneurysm and dissection with contraindications for surgical management. We also discuss the significant risk that the patient had to overcome to survive an undiagnosed aortic dissection and an acute ST elevation myocardial infarction. PMID:27757405

  18. Dissection Videos Do Not Improve Anatomy Examination Scores

    ERIC Educational Resources Information Center

    Mahmud, Waqas; Hyder, Omar; Butt, Jamaal; Aftab, Arsalan

    2011-01-01

    In this quasi-experimental study, we describe the effect of showing dissection videos on first-year medical students' performance in terms of test scores during a gross anatomy course. We also surveyed students' perception regarding the showing of dissection videos. Two hundred eighty-seven first-year medical students at Rawalpindi Medical College…

  19. Outcomes of a Rotational Dissection System in Gross Anatomy

    ERIC Educational Resources Information Center

    Marshak, David W.; Oakes, Joanne; Hsieh, Pei-Hsuan; Chuang, Alice Z.; Cleary, Leonard J.

    2015-01-01

    At the University of Texas Houston Medical School, a rotational dissection system was introduced to improve coordination between the Gross Anatomy and the Introduction to Clinical Medicine (ICM) courses. Six students were assigned to each cadaver and divided into two teams. For each laboratory, one team was assigned to dissect and the other to…

  20. Student Attitudes toward Cadaveric Dissection at a UK Medical School

    ERIC Educational Resources Information Center

    Quince, Thelma A.; Barclay, Stephen I. G.; Spear, Michelle; Parker, Richard A.; Wood, Diana F.

    2011-01-01

    A more humanistic approach toward dissection has emerged. However, student attitudes toward this approach are unknown and the influences on such attitudes are little understood. One hundred and fifty-six first-year medical students participated in a study examining firstly, attitudes toward the process of dissection and the personhood of the…

  1. Internal carotid artery dissection associated with scuba diving.

    PubMed

    Nelson, E E

    1995-01-01

    A 52-year-old man presented to the emergency department with dysphasia and a headache after scuba diving. He was treated initially for decompression sickness. Subsequent workup revealed bilateral internal carotid artery dissection. The risk factors, presenting symptoms, diagnosis, and treatment of internal carotid artery dissection are reviewed. The importance of considering unusual causes of neurologic deficits after scuba diving is emphasized.

  2. A Modified Dissection Method to Preserve Neck Structures

    ERIC Educational Resources Information Center

    Hankin, Mark H.; Stoller, Jeremy L.

    2009-01-01

    The neck is not only one of the more challenging anatomical regions to dissect but also has important application to clinical conditions, diseases, and procedures. In this study, we describe two simple modifications for dissection of the neck that (1) aid in the identification and preservation of the cutaneous branches of the cervical plexus and…

  3. Traditional versus Computer-Based Dissections in Enhancing Learning in a Tertiary Setting: A Student Perspective.

    ERIC Educational Resources Information Center

    Franklin, Sue; Peat, Mary; Lewis, Alison

    2002-01-01

    Describes a study that investigates both the use and usefulness of laboratory dissections and computer-based dissections in a tertiary, first-year human biology course. Explores attitudes toward dissection. (DDR)

  4. CrossFit-related cervical internal carotid artery dissection.

    PubMed

    Lu, Albert; Shen, Peter; Lee, Paul; Dahlin, Brian; Waldau, Ben; Nidecker, Anna E; Nundkumar, Anoop; Bobinski, Matthew

    2015-08-01

    CrossFit is a high-intensity strength and conditioning program that has gained popularity over the past decade. Potential injuries associated with CrossFit training have been suggested in past reports. We report three cases of cervical carotid dissection that are associated with CrossFit workouts. Patient 1 suffered a distal cervical internal carotid artery (ICA) dissection near the skull base and a small infarct in Wernicke's area. He was placed on anticoagulation and on follow-up has near complete recovery. Patient 2 suffered a proximal cervical ICA dissection that led to arterial occlusion and recurrent middle cerebral artery territory infarcts and significant neurological sequelae. Patient 3 had a skull base ICA dissection that led to a partial Horner's syndrome but no cerebral infarct. While direct causality cannot be proven, intense CrossFit workouts may have led to the ICA dissections in these patients. PMID:25917634

  5. CrossFit-related cervical internal carotid artery dissection.

    PubMed

    Lu, Albert; Shen, Peter; Lee, Paul; Dahlin, Brian; Waldau, Ben; Nidecker, Anna E; Nundkumar, Anoop; Bobinski, Matthew

    2015-08-01

    CrossFit is a high-intensity strength and conditioning program that has gained popularity over the past decade. Potential injuries associated with CrossFit training have been suggested in past reports. We report three cases of cervical carotid dissection that are associated with CrossFit workouts. Patient 1 suffered a distal cervical internal carotid artery (ICA) dissection near the skull base and a small infarct in Wernicke's area. He was placed on anticoagulation and on follow-up has near complete recovery. Patient 2 suffered a proximal cervical ICA dissection that led to arterial occlusion and recurrent middle cerebral artery territory infarcts and significant neurological sequelae. Patient 3 had a skull base ICA dissection that led to a partial Horner's syndrome but no cerebral infarct. While direct causality cannot be proven, intense CrossFit workouts may have led to the ICA dissections in these patients.

  6. Spontaneous left main dissection treated by percutaneous coronary intervention.

    PubMed

    Sousa, Alexandra; Lopes, Ricardo; Silva, João Carlos; Maciel, M Júlia

    2013-06-01

    Spontaneous coronary artery dissection is a rare cause of acute coronary events or sudden cardiac death. The clinical presentation is highly variable and prognosis varies widely, depending mainly on how rapidly it is diagnosed. Prompt treatment is also essential, and includes medical management, percutaneous coronary intervention and surgical revascularization. We describe the case of a young woman presenting with spontaneous coronary artery dissection of the left main coronary artery, first diagnosed as coronary thrombus, who underwent successful percutaneous coronary stenting. This report highlights the need to include spontaneous coronary artery dissection in differential diagnosis of chest pain in young women and that distinguishing between coronary thrombus and coronary artery dissection is not always straightforward. To our knowledge this is the fourth case of left main stenting in a patient with spontaneous coronary artery dissection described in the literature.

  7. Common carotid artery dissection caused by radiotherapy: A case report

    PubMed Central

    Wang, Jiayan; Yue, Dandan; Chen, Xin; Wei, Zhenyu; Lu, Wenmei; Wu, Danhong

    2016-01-01

    In the present study, a case of acute cerebral infarction with radiation-induced carotid artery dissection is reported. Carotid artery dissection is generally asymptomatic at the early stages. Due to the non-specific clinical manifestations of carotid artery dissection, a detailed inquiry of the past history of a patient has a critical role in making a diagnosis of radiation-induced common carotid artery dissection. Onset of acute ischemic stroke is the predominant manifestation, and for patients with a history of head-and-neck radiotherapy, dissection should be considered. The condition may progress rapidly, and result in a poor prognosis. Therefore, a correct early diagnosis and initiation of appropriate therapy may lead to rapid recovery, and influence the overall prognosis.

  8. Dissecting aneurysm of the anterior temporal artery: case report.

    PubMed

    Umeoka, Katsuya; Shirokane, Kazutaka; Mizunari, Takayuki; Kobayashi, Shiro; Teramoto, Akira

    2011-01-01

    A 65-year-old woman presented a rare dissecting aneurysm of the anterior temporal artery (ATA) manifesting as headache. Computed tomography and magnetic resonance imaging revealed a mixed-density mass in the horizontal segment of the middle cerebral artery. Emergent angiography demonstrated aneurysmal dilatation and a thrombosed mass in the sylvian fissure. Infectious aneurysm was excluded. She underwent emergent surgery to reduce the risk of repeated infarction and hemorrhage. The distal side of the ATA manifested occlusive changes suggestive of arterial dissection. The proximal side of the ATA was ligated and the lesion was excised. Histological examination confirmed that the aneurysmal dilatation was attributable to arterial dissection due to disruption of the internal elastic lamina. Distal dissecting aneurysms may occur in the absence of infectious disease. We recommend that ruptured distal dissecting aneurysms be treated surgically in the acute stage immediately after detection.

  9. Common carotid artery dissection caused by radiotherapy: A case report

    PubMed Central

    Wang, Jiayan; Yue, Dandan; Chen, Xin; Wei, Zhenyu; Lu, Wenmei; Wu, Danhong

    2016-01-01

    In the present study, a case of acute cerebral infarction with radiation-induced carotid artery dissection is reported. Carotid artery dissection is generally asymptomatic at the early stages. Due to the non-specific clinical manifestations of carotid artery dissection, a detailed inquiry of the past history of a patient has a critical role in making a diagnosis of radiation-induced common carotid artery dissection. Onset of acute ischemic stroke is the predominant manifestation, and for patients with a history of head-and-neck radiotherapy, dissection should be considered. The condition may progress rapidly, and result in a poor prognosis. Therefore, a correct early diagnosis and initiation of appropriate therapy may lead to rapid recovery, and influence the overall prognosis. PMID:27699045

  10. Comparative phosphoproteome analysis of the developing grains in bread wheat (Triticum aestivum L.) under well-watered and water-deficit conditions.

    PubMed

    Zhang, Ming; Ma, Cao-Ying; Lv, Dong-Wen; Zhen, Shou-Min; Li, Xiao-Hui; Yan, Yue-Ming

    2014-10-01

    Wheat (Triticum aestivum), one of the most important cereal crops, is often threatened by drought. In this study, water deficit significantly reduced the height of plants and yield of grains. To explore further the effect of drought stress on the development and yield of grains, we first performed a large scale phosphoproteome analysis of developing grains in wheat. A total of 590 unique phosphopeptides, representing 471 phosphoproteins, were identified under well-watered conditions. Motif-X analysis showed that four motifs were enriched, including [sP], [Rxxs], [sDxE], and [sxD]. Through comparative phosphoproteome analysis between well-watered and water-deficit conditions, we found that 63 unique phosphopeptides, corresponding to 61 phosphoproteins, showed significant changes in phosphorylation level (≥2-fold intensities). Functional analysis suggested that some of these proteins may be involved in signal transduction, embryo and endosperm development of grains, and drought response and defense under water-deficit conditions. Moreover, we also found that some chaperones may play important roles in protein refolding or degradation when the plant is subjected to water stress. These results provide a detailed insight into the stress response and defense mechanisms of developmental grains at the phosphoproteome level. They also suggested some potential candidates for further study of transgenosis and drought stress as well as incorporation into molecular breeding for drought resistance.

  11. Infected Aneurysm after Endoscopic Submucosal Dissection.

    PubMed

    Gen, Shiko; Usui, Ryuichi; Sasaki, Takaya; Nobe, Kanako; Takahashi, Aya; Okudaira, Keisuke; Ikeda, Naofumi

    2016-01-01

    A 79-year-old man on hemodialysis was hospitalized for further investigation. Early gastric cancer was diagnosed by gastrointestinal endoscopy and endoscopic submucosal dissection (ESD) was performed. Fever and abdominal pain thereafter developed, and a severe inflammatory response was observed on a blood test. Contrast computed tomography (CT) showed ulcer-like projections and soft tissue surrounding the aorta, from the celiac to left renal artery. An infected aneurysm was diagnosed. Although infected aneurysms developing after laparoscopic cholecystectomy or biopsy of contiguous esophageal duplication cyst have been reported, those developing after ESD have not. When fever and abdominal pain develop after ESD, an infected aneurysm should be considered and contrast CT performed. PMID:27181541

  12. Spontaneous renal artery dissection with renal infarction.

    PubMed

    Renaud, Sophie; Leray-Moraguès, Hélène; Chenine, Leila; Canaud, Ludovic; Vernhet-Kovacsik, Hélène; Canaud, Bernard

    2012-06-01

    Spontaneous renal artery dissection (SRAD) is a rare entity, which often presents diagnostic difficulties because of its non-specific clinical presentation. We report six cases complicated with renal infarction, occurring in middle-aged male patients without risk factors, illustrating the difficulty and delay for diagnosing SRAD. Ultrasound and Doppler imaging were not sensitive enough to confirm the diagnosis, and contrast-enhanced abdominal computed tomography was used to correct the diagnosis and allow the clinicians to propose appropriate treatment. We conclude that considering the urgency in diagnosing and treating SRAD, contrast enhanced abdominal tomography and/or abdominal magnetic resonance imaging should be proposed as soon as a suspicion of SRAD is evoked by the clinical presentation.

  13. Recent traction methods for endoscopic submucosal dissection

    PubMed Central

    Tsuji, Kunihiro; Yoshida, Naohiro; Nakanishi, Hiroyoshi; Takemura, Kenichi; Yamada, Shinya; Doyama, Hisashi

    2016-01-01

    Endoscopic mucosal resection (EMR) is problematic with regard to en bloc and curable resection rates. Advancements in endoscopic techniques have enabled novel endoscopic approaches such as endoscopic submucosal dissection (ESD), which has overcome some EMR problems, and has become the standard treatment for gastrointestinal tumors. However, ESD is technically difficult. Procedure time is longer and complications such as intraoperative perforation and bleeding occur more frequently than in EMR. Recently various traction methods have been introduced to facilitate ESD procedures, such as clip with line, external forceps, clip and snare, internal traction, double scope, and magnetic anchor. Each method must be used appropriately according to the anatomical characteristics. In this review we discuss recently proposed traction methods for ESD based on the characteristics of various anatomical sites. PMID:27468186

  14. Endoscopic submucosal dissection for colorectal neoplasms

    PubMed Central

    Takamaru, Hiroyuki; Mori, Genki; Yamada, Masayoshi; Kinjo, Yuzuru; So, Eriko; Abe, Seiichiro; Otake, Yosuke; Nakajima, Takeshi; Matsuda, Takahisa; Saito, Yutaka

    2014-01-01

    Endoscopic submucosal dissection (ESD) is an established therapeutic technique for the treatment of gastrointestinal neoplasms. Because it is typically completed as en bloc resection, this technique provides a complete specimen for precise pathological evaluation. On the other hand, ESD is not as widely applied in treating colorectal neoplasms as with gastric cancers, due to its technical difficulty, longer procedure time, and increased risk of perforation. However, some devices that facilitate ESD and improve the safety of the procedure have been recently reported, and the use of the technique has gradually spread worldwide. Endoscopists who begin to perform ESD need to recognize the indications of ESD, the technical issue involved in this procedure, and its associated complications. This review outlines the methods and certain types of devices used for colorectal ESD. PMID:25333002

  15. Dissecting Japan's Dengue Outbreak in 2014

    PubMed Central

    Quam, Mikkel B.; Sessions, October; Kamaraj, Uma Sangumathi; Rocklöv, Joacim; Wilder-Smith, Annelies

    2016-01-01

    Despite Japan's temperate climate, a dengue outbreak occurred in Tokyo for the first time in over 70 years in 2014. We dissected this dengue outbreak based on phylogenetic analysis, travel interconnectivity, and environmental drivers for dengue epidemics. Comparing the available dengue virus 1 (DENV1) E gene sequence from this outbreak with 3,282 unique DENV1 sequences in National Center for Biotechnology Information suggested that the DENV might have been imported from China, Indonesia, Singapore, or Vietnam. With travelers arriving into Japan, Guangzhou (China) may have been the source of DENV introduction, given that Guangzhou also reported a large-scale dengue outbreak in 2014. Coinciding with the 2014 outbreak, Tokyo's climate conditions permitted the amplification of Aedes vectors and the annual peak of vectorial capacity. Given suitable vectors and climate conditions in addition to increasing interconnectivity with endemic areas of Asia, Tokyo's 2014 outbreak did not come as a surprise and may foretell more to come. PMID:26711518

  16. Surgical Management of Intracranial Artery Dissection

    PubMed Central

    ARIMURA, Koichi; IIHARA, Koji

    2016-01-01

    Intracranial artery dissection (IAD) is a relatively rare cause of stroke, but it has been recognized increasingly with recent advances of the neuroimaging technique. Since rebleeding occurs frequently in the acute stage in the ruptured IAD, urgent surgical treatment should be performed to prevent rebleeding. On the other hand, surgical treatment for unruptured IAD is controversial because it has little risk for bleeding. However, surgical treatment for unruptured IAD may be considered if the formation or enlargement of the aneurysmal dilatation has been confirmed. Since there are several proposed surgical strategies for IAD, it is important to select an appropriate strategy on a case-by-case basis. If the risk of infarction due to vessel occlusion is high, combined bypass surgery should be considered. PMID:27063145

  17. Dissecting Japan's Dengue Outbreak in 2014.

    PubMed

    Quam, Mikkel B; Sessions, October; Kamaraj, Uma Sangumathi; Rocklöv, Joacim; Wilder-Smith, Annelies

    2016-02-01

    Despite Japan's temperate climate, a dengue outbreak occurred in Tokyo for the first time in over 70 years in 2014. We dissected this dengue outbreak based on phylogenetic analysis, travel interconnectivity, and environmental drivers for dengue epidemics. Comparing the available dengue virus 1 (DENV1) E gene sequence from this outbreak with 3,282 unique DENV1 sequences in National Center for Biotechnology Information suggested that the DENV might have been imported from China, Indonesia, Singapore, or Vietnam. With travelers arriving into Japan, Guangzhou (China) may have been the source of DENV introduction, given that Guangzhou also reported a large-scale dengue outbreak in 2014. Coinciding with the 2014 outbreak, Tokyo's climate conditions permitted the amplification of Aedes vectors and the annual peak of vectorial capacity. Given suitable vectors and climate conditions in addition to increasing interconnectivity with endemic areas of Asia, Tokyo's 2014 outbreak did not come as a surprise and may foretell more to come.

  18. A variant of nested dissection for solving n by n grid problems

    NASA Technical Reports Server (NTRS)

    George, A.; Poole, W. G., Jr.; Voigt, R. G.

    1976-01-01

    Nested dissection orderings are known to be very effective for solving the sparse positive definite linear systems which arise from n by n grid problems. In this paper nested dissection is shown to be the final step of incomplete nested dissection, an ordering which corresponds to the premature termination of dissection. Analyses of the arithmetic and storage requirements for incomplete nested dissection are given, and the ordering is shown to be competitive with nested dissection under certain conditions.

  19. Influence of a Dissection Video Clip on Anxiety, Affect, and Self-Efficacy in Educational Dissection: A Treatment Study

    ERIC Educational Resources Information Center

    Randler, Christoph; Demirhan, Eda; Wüst-Ackermann, Peter; Desch, Inga H.

    2016-01-01

    In science education, dissections of animals are an integral part of teaching, but they often evoke negative emotions. We aimed at reducing negative emotions (anxiety, negative affect [NA]) and increasing positive affect (PA) and self-efficacy by an experimental intervention using a predissection video to instruct students about fish dissection.…

  20. The Effects of Computer Animated Dissection versus Preserved Animal Dissection on the Student Achievement in a High School Biology Class.

    ERIC Educational Resources Information Center

    Kariuki, Patrick; Paulson, Ronda

    The purpose of this study was to examine the effectiveness of computer-animated dissection techniques versus the effectiveness of traditional dissection techniques as related to student achievement. The sample used was 104 general biology students from a small, rural high school in Northeast Tennessee. Random selection was used to separate the…

  1. Influence of a Dissection Video Clip on Anxiety, Affect, and Self-Efficacy in Educational Dissection: A Treatment Study

    PubMed Central

    Randler, Christoph; Demirhan, Eda; Wüst-Ackermann, Peter; Desch, Inga H.

    2016-01-01

    In science education, dissections of animals are an integral part of teaching, but they often evoke negative emotions. We aimed at reducing negative emotions (anxiety, negative affect [NA]) and increasing positive affect (PA) and self-efficacy by an experimental intervention using a predissection video to instruct students about fish dissection. We compared this treatment with another group that watched a life history video about the fish. The participants were 135 students studying to become biology teachers. Seventy received the treatment with the dissection video, and 65 viewed the life history video. We applied a pre/posttest treatment-comparison design and used the Positive and Negative Affect Schedule (PANAS), the State–Trait–Anxiety Inventory for State (STAI-S), and a self-efficacy measure three times: before the lesson (pretest), after the film treatment (posttest 1), and after the dissection (posttest 2). The dissection film group scored higher in PA, NA, and state anxiety (STAI-S) after the dissection video treatment and higher in self-efficacy after the dissection. The life history group showed no differences between the pretest and posttest 1. The dissection film has clear benefits—increasing PA and self-efficacy—that come at the cost of higher NA and higher STAI-S. PMID:27290738

  2. Painless Type B Aortic Dissection: Insights From the International Registry of Acute Aortic Dissection

    PubMed Central

    Tolenaar, Jip L.; Hutchison, Stuart J.; Montgomery, Dan; O'Gara, Patrick; Fattori, Rosella; Pyeritz, Reed E.; Pape, Linda; Suzuki, Toru; Evangelista, Arturo; Moll, Frans L.; Rampoldi, Vincenzo; Isselbacher, Eric M.; Nienaber, Cristoph A.; Eagle, Kim A.; Trimarchi, Santi

    2013-01-01

    Introduction: The classical presentation of a patient with Type B acute aortic dissection (TBAAD) is characterized by severe chest, back, or abdominal pain, ripping or tearing in nature. However, some patients present with painless acute aortic dissection, which can lead to a delay in diagnosis and treatment. We utilized the International Registry on Acute Aortic Dissections (IRAD) database to study these patients. Methods: We analyzed 43 painless TBAAD patients enrolled in the database between January 1996 and July 2012. The differences in presentation, diagnostics, management, and outcome were compared with patients presenting with painful TBAAD. Results: Among the 1162 TBAAD patients enrolled in IRAD, 43 patients presented with painless TBAAD (3.7%). The mean age of patients with painless TBAAD was significantly higher than normal TBAAD patients (69.2 versus 63.3 years, P = 0.020). The presence of atherosclerosis (46.4% versus 30.1%, P = 0.022), diabetes (17.9% versus 7.5%; P = 0.018), and other aortic diseases (8.6% versus 2.3%, P= 0.051), such as prior aortic aneurysm (31% versus 18.8% P = 0.049) was more common in these patients. Median delay time between presentation and diagnosis was longer in painless patients (median 34.0 versus 19.0 hours; P = 0.006). Dissection of iatrogenic origin (19.5% versus 1.3%; P < 0.001) was significantly more frequent in the painless group. The in-hospital mortality was 18.6% in the painless group, compared with an in-hospital mortality of 9.9% in the control group (P = 0.063). Conclusion: Painless TBAAD is a relatively rare presentation (3.7%) of aortic dissection, and is often associated with a history of atherosclerosis, diabetes, prior aortic disease including aortic aneurysm, and an iatrogenic origin. We observed a trend for increased in-hospital mortality in painless TBAAD patients, which may be the result of a delay in diagnosis and management. Therefore, physicians should be aware of this relative rare presentation of

  3. Cranial nerve palsies in spontaneous carotid artery dissection.

    PubMed Central

    Sturzenegger, M; Huber, P

    1993-01-01

    Two patients had isolated unilateral cranial nerve palsies due to spontaneous internal carotid artery (ICA) dissection without ischaemic cerebral involvement. One had acute glossopharyngeal and vagal, the other isolated hypoglossal nerve palsy. Reviewing all reported cases of angiographically confirmed ICA dissection in the literature, 36 additional cases with unequivocal ipsilateral cranial nerve palsies were analysed. While an isolated palsy of the IXth and Xth has not been reported previously, palsies of the XIIth nerve or the IXth to XIIth nerves were most frequently found. In these patients, lower cranial nerve palsies are probably the result of compression by an enlarging ICA due to mural haematoma. Symptoms and signs indicative of carotid dissection were concurrently present only in some reported cases. This raises the question of unrecognised carotid dissection as a cause of isolated cranial nerve palsies. When the dissection occurs in the subadventitial layer without relevant narrowing of the arterial lumen and when an aneurysm is thrombosed, angiography does not reliably yield the diagnosis. Therefore, carotid dissection might have been underestimated as a cause of isolated lower cranial nerve palsies before the advent of MRI. MRI demonstrates directly the extension of the wall haematoma in the axial and longitudinal planes. Some arteriopathies such as fibromuscular dysplasia and tortuosity make a vessel predisposed to dissection. Images PMID:8229030

  4. Acute Type A Aortic Dissection Missed as Acute Coronary Syndrome

    PubMed Central

    Ansari-Ramandi, Mohammad Mostafa; Firoozi, Ata

    2016-01-01

    Although the aortic dissection is not common, its outcome is frequently fatal, and many patients with aortic dissection die before referral to the hospital or any diagnostic testing. The symptoms of aortic dissection can be similar to myocardial ischemia. A 66-year-old male was referred to our hospital with suspicion of aortic dissection after echocardiography done for evaluating his high blood pressure. He had symptoms of acute coronary syndrome two years before and had done coronary angiography. On presentation to our hospital he had a high blood pressure. On reviewing his past medical history and examining, in the film of coronary angiography, the dissection flap in ascending aorta was identified. Although type A aortic dissection is a catastrophic condition with high mortality and requires prompt surgical treatment but in some cases it may be misdiagnosed as acute coronary syndrome. Sometimes against its high mortality when left untreated, patients survive and are diagnosed later in life incidentally. So it is of great importance to have great clinical suspicion for aortic dissection in patients referring to the hospital with chest pain and the predisposing factors. PMID:27437290

  5. Evaluating Multiplexed Quantitative Phosphopeptide Analysis on a Hybrid Quadrupole Mass Filter/Linear Ion Trap/Orbitrap Mass Spectrometer

    PubMed Central

    2015-01-01

    As a driver for many biological processes, phosphorylation remains an area of intense research interest. Advances in multiplexed quantitation utilizing isobaric tags (e.g., TMT and iTRAQ) have the potential to create a new paradigm in quantitative proteomics. New instrumentation and software are propelling these multiplexed workflows forward, which results in more accurate, sensitive, and reproducible quantitation across tens of thousands of phosphopeptides. This study assesses the performance of multiplexed quantitative phosphoproteomics on the Orbitrap Fusion mass spectrometer. Utilizing a two-phosphoproteome model of precursor ion interference, we assessed the accuracy of phosphopeptide quantitation across a variety of experimental approaches. These methods included the use of synchronous precursor selection (SPS) to enhance TMT reporter ion intensity and accuracy. We found that (i) ratio distortion remained a problem for phosphopeptide analysis in multiplexed quantitative workflows, (ii) ratio distortion can be overcome by the use of an SPS-MS3 scan, (iii) interfering ions generally possessed a different charge state than the target precursor, and (iv) selecting only the phosphate neutral loss peak (single notch) for the MS3 scan still provided accurate ratio measurements. Remarkably, these data suggest that the underlying cause of interference may not be due to coeluting and cofragmented peptides but instead from consistent, low level background fragmentation. Finally, as a proof-of-concept 10-plex experiment, we compared phosphopeptide levels from five murine brains to five livers. In total, the SPS-MS3 method quantified 38 247 phosphopeptides, corresponding to 11 000 phosphorylation sites. With 10 measurements recorded for each phosphopeptide, this equates to more than 628 000 binary comparisons collected in less than 48 h. PMID:25521595

  6. Nearly Asymptomatic Eight-Month Thoracic Aortic Dissection

    PubMed Central

    Kumar, Arjun; Kumar, Krishan; Zeltser, Roman; Makaryus, Amgad N.

    2016-01-01

    Thoracic aortic dissection is a rare, but lethal, medical condition that is either misdiagnosed as a myocardial infarction or overlooked completely. Though thoracic aortic dissections are commonly diagnosed in patients exhibiting sharp chest pain, there are some notable cases where patients do not report the expected severity of pain. We report a unique case of a patient with a thoracic aortic dissection who was initially nearly asymptomatic for eight months, in order to heighten awareness, highlight diagnosis protocol, and improve prognosis for this commonly misdiagnosed, but fatal, condition. PMID:27257400

  7. Abdominal aortic grafting for spontaneous infrarenal abdominal aortic dissection.

    PubMed

    Iwasaki, Hiroto; Shibuya, Takashi; Shintani, Takashi; Uenaka, Hisazumi; Suehiro, Shigefumi; Satoh, Hisashi

    2010-02-01

    This case report concerns a 62-year-old woman with spontaneous infrarenal abdominal aortic dissection, which developed into claudication and rest pain in the lower extremity. Multi-row detector computed tomography showed the entry site of the abdominal aortic dissection at the second lumbar artery, while the reentry site was found intraoperatively at the median sacral artery, indicating that the false lumen had progressed and compressed the true lumen. A direct approach involving grafting appears to be an effective procedure for resolving mesenteric and lower extremity hypoperfusion due to aortic dissection with a dilated false channel, even during the acute period. PMID:19879731

  8. Aortic dissection accompanied by preeclampsia in a postpartum young woman

    PubMed Central

    Park, Jin-Wan; Kim, Su-Mi; Yu, Gyu-Bong

    2016-01-01

    Aortic dissection is very rare in obstetrics, but it is a fatal disease. A 37-weeks primigravida woman with dyspnea and pitting edema presented to our emergency room. The patient was diagnosed with preeclampsia and underwent an emergency cesarean section under spinal anesthesia. The patient complained of severe dyspnea after the cesarean section, and the chest computed tomography scan was done. With the finding of aortic dissection, cardiopulmonary arrest occurred 5 hours after the cesarean section, and the patient died without reaction to cardio-pulmonary resuscitation. If a patient with preeclampsia complains of severe dyspnea or chest pain, aortic dissection needs to be suspected and a diagnosis should not be delayed.

  9. Pediatric traumatic carotid, vertebral and cerebral artery dissections: a review.

    PubMed

    Mortazavi, Martin M; Verma, Ketan; Tubbs, R Shane; Harrigan, Mark

    2011-12-01

    Traumatic cerebral dissections are rare but potentially dangerous conditions that through improved diagnostics have recently gained increased interest. However, there is still a significant lack of knowledge on the natural history, as well as on the best treatment options. Most of the literature on this topic consists of case reports and retrospective studies with no prospective randomized controlled studies. In our review, we highlight the fact that there is no level 1 evidence for the natural history of cerebral dissections or for the best treatment. We present 26 case studies derived from 70 pediatric patients affected by dissections, occlusions, and pseudoaneurysms.

  10. Fenestrated internal jugular vein: a rare finding in neck dissection.

    PubMed

    Moreno-Sánchez, M; Hernández Vila, C; González-García, R; Monje, F

    2015-09-01

    Fenestration of the internal jugular vein (IJV) is an extremely rare phenomenon. An 85-year-old woman was referred to our department with T2N0M0 squamous cell carcinoma of the right floor of the mouth. The patient underwent local resection, an ipsilateral modified type 3 radical neck dissection, and microsurgical reconstruction. During neck dissection, a fenestration was observed in the middle of the IJV. No structure passed through the fenestration. Such anatomical variations are rare, but clinicians and surgeons should be aware of them in order to avoid damage to the important vascular structures, especially if a neck dissection is performed.

  11. Neck dissection with cervical sensory preservation in thyroid cancer.

    PubMed

    Xue, Shuai; Wang, Peisong; Chen, Guang

    2013-11-01

    Thyroid cancer is the most common endocrine malignancy. Recently, controversy has focused on the management of lymph node metastases, which represent approximately 90% of disease recurrences and may require considerable time, effort, and resources to diagnose and treat. Neck dissections play an essential role in the management of head and neck cancer. A modified radical neck dissection (MND) refers to resection of the lymph nodes in levels II through V and often including the central nodes in level VI. When performing modified neck dissection, we recommend to protect more reserved cervical plexus. The purpose is to better protect patient's neck skin feeling.

  12. Spontaneous renal artery dissection complicating with renal infarction.

    PubMed

    Tsai, Tsung-Han; Su, Jung-Tsung; Hu, Sung-Yuan; Chao, Chih-Chung; Tsan, Yu-Tse; Lin, Tzu-Chieh

    2010-12-01

    Spontaneous renal artery dissection (SRAD) is a rare entity. We reported a 30-year-old healthy man presenting with sudden onset of left flank pain. Abdominal plain film and sonography were unremarkable. The contrast-enhanced abdominal computed tomographic (CT) scan demonstrated a dissecting intimal flap of the left distal renal artery (RA) complicating infarction. Selective angiography of the renal artery disclosed a long dissection of left distal RA with a patent true lumen and occlusion of left accessory RA. Conservative treatment with control of blood pressure and antiplatelet agent was prescribed. The patient was discharged with an uneventful condition on day 5.

  13. Optogenetic dissection of medial prefrontal cortex circuitry

    PubMed Central

    Riga, Danai; Matos, Mariana R.; Glas, Annet; Smit, August B.; Spijker, Sabine; Van den Oever, Michel C.

    2014-01-01

    The medial prefrontal cortex (mPFC) is critically involved in numerous cognitive functions, including attention, inhibitory control, habit formation, working memory and long-term memory. Moreover, through its dense interconnectivity with subcortical regions (e.g., thalamus, striatum, amygdala and hippocampus), the mPFC is thought to exert top-down executive control over the processing of aversive and appetitive stimuli. Because the mPFC has been implicated in the processing of a wide range of cognitive and emotional stimuli, it is thought to function as a central hub in the brain circuitry mediating symptoms of psychiatric disorders. New optogenetics technology enables anatomical and functional dissection of mPFC circuitry with unprecedented spatial and temporal resolution. This provides important novel insights in the contribution of specific neuronal subpopulations and their connectivity to mPFC function in health and disease states. In this review, we present the current knowledge obtained with optogenetic methods concerning mPFC function and dysfunction and integrate this with findings from traditional intervention approaches used to investigate the mPFC circuitry in animal models of cognitive processing and psychiatric disorders. PMID:25538574

  14. Esophageal Stricture Prevention after Endoscopic Submucosal Dissection

    PubMed Central

    Jain, Deepanshu; Singhal, Shashideep

    2016-01-01

    Advances in diagnostic modalities and improvement in surveillance programs for Barrett esophagus has resulted in an increase in the incidence of superficial esophageal cancers (SECs). SEC, due to their limited metastatic potential, are amenable to non-invasive treatment modalities. Endoscopic ultrasound, endoscopic mucosal resection, and endoscopic submucosal dissection (ESD) are some of the new modalities that gastroenterologists have used over the last decade to diagnose and treat SEC. However, esophageal stricture (ES) is a very common complication and a major cause of morbidity post-ESD. In the past few years, there has been a tremendous effort to reduce the incidence of ES among patients undergoing ESD. Steroids have shown the most consistent results over time with minimal complications although the preferred mode of delivery is debatable, with both systemic and local therapy having pros and cons for specific subgroups of patients. Newer modalities such as esophageal stents, autologous cell sheet transplantation, polyglycolic acid, and tranilast have shown promising results but the depth of experience with these methods is still limited. We have summarized case reports, prospective single center studies, and randomized controlled trials describing the various methods intended to reduce the incidence of ES after ESD. Indications, techniques, outcomes, limitations, and reported complications are discussed. PMID:26949124

  15. Genetic dissection of drug resistance in trypanosomes.

    PubMed

    Alsford, Sam; Kelly, John M; Baker, Nicola; Horn, David

    2013-10-01

    The trypanosomes cause two neglected tropical diseases, Chagas disease in the Americas and African trypanosomiasis in sub-Saharan Africa. Over recent years a raft of molecular tools have been developed enabling the genetic dissection of many aspects of trypanosome biology, including the mechanisms underlying resistance to some of the current clinical and veterinary drugs. This has led to the identification and characterization of key resistance determinants, including transporters for the anti-Trypanosoma brucei drugs, melarsoprol, pentamidine and eflornithine, and the activator of nifurtimox-benznidazole, the anti-Trypanosoma cruzi drugs. More recently, advances in sequencing technology, combined with the development of RNA interference libraries in the clinically relevant bloodstream form of T. brucei have led to an exponential increase in the number of proteins known to interact either directly or indirectly with the anti-trypanosomal drugs. In this review, we discuss these findings and the technological developments that are set to further revolutionise our understanding of drug-trypanosome interactions. The new knowledge gained should inform the development of novel interventions against the devastating diseases caused by these parasites.

  16. Paediatric tonsillectomy: radiofrequency-based plasma dissection compared to cold dissection with sutures

    PubMed Central

    Di Rienzo Businco, L; Coen Tirelli, G

    2008-01-01

    Summary Aim of this study was to compare post-operative recovery over 14 days in children submitted to tonsillectomy using a bipolar radiofrequency-based plasma device (Coblation®, Evac 70, ArthroCare Corp, Sunnyvale, CA, USA) to cold dissection. Paediatric patients (n = 42) aged 5-16 years old with chronic tonsillitis underwent tonsillectomy using cold dissection with suture ligatures or a plasma device (Evac 70, ArthroCare Corp, Sunnyvale, CA, USA). Pain intensity on the first day, use of analgesics, type of diet, and days of pain, fever, nausea, and absence from school were determined. Groups were compared using time-to-event (Kaplan-Meier) curves and statistically evaluated using the Breslow (generalized Wilcoxon) test. Children undergoing plasma tonsillectomy reported significantly less pain on the first post-operative day (1.2 ± 0.9 vs. 3.5 ± 1.5, p < 0.001), fewer days of pain (4.8 ± 1.5 vs. 9.4 ± 1.2, p < 0.001), pain medication withdrawal (2.6 ± 1.3 vs. 4.5 ± 1.3, p < 0.001) and earlier use of liquid diet (5.1 ± 1.4 vs. 8.5 ± 2.1, p < 0.001), and fewer school days lost (5.3 ± 1.7 vs. 8.9 ± 1.5, p < 0.001). After completing this study, plasma tonsillectomy was adopted for the majority of cases. Benefits of the plasma device include the possibility both to excise tissue and coagulate bleeding vessels using the same device whilst improving quality of post-operative recovery over cold dissection with suture ligatures. PMID:18669070

  17. Non-invasive diagnosis of internal carotid artery dissections.

    PubMed Central

    Müllges, W; Ringelstein, E B; Leibold, M

    1992-01-01

    Arteriography is thought to be mandatory for the diagnosis of internal carotid artery (ICA) dissection. With the introduction of transcranial Doppler sonography (TCD) and magnetic resonance imaging (MRI), however, this is no longer the case. In 13 consecutive patients with ICA dissections the diagnosis was made by means of non-invasive tests including extracranial and transcranial Doppler sonography, contrast enhanced computed tomography (ceCT), and, in five patients, MRI. Intra-arterial digital subtraction angiography used as the gold standard in all cases was confirmative. Extracranial and transcranial ultrasound findings indicative of the diagnosis could be identified. MRI directly demonstrated the intramural haematoma and the false lumen of the dissected artery. These non-invasive techniques also allowed for repetitive follow up examinations. They were, however, unable to demonstrate false aneurysms in the chronic state. Results show that the diagnosis of carotid dissection can be made by means of cerebrovascular ultrasound and MRI. Images PMID:1538235

  18. Acute and chronic acalculous cholecystitis associated with aortic dissection.

    PubMed

    Inagaki, Fuyuki F; Hara, Yoshiaki; Kamei, Masako; Tanaka, Michio; Yasuno, Masamichi

    2015-01-01

    Acalculous cholecystitis is a rare but life-threatening disease, but its pathogenesis is not fully revealed yet. We experienced two acalculous cholecystitis cases associated with aortic dissection. In Case 1, acalculous cholecystitis occurred just after the exacerbation of the aortic dissection. Laparotomy showed necrotized cholecystitis with fresh thrombi formation. Case 2 developed acalculous cholecystitis on the 65th hospital day of aortic dissection. Laparotomy revealed the perforation of the gallbladder. Histological study revealed fibrosis and hemosiderosis in the subserosal layer. The histological findings of these two patients are quite different: Case 1 is acute ischemic and Case 2 is chronic ischemic. While a few cases of acute ischemic cholecystitis have been reported previously, chronic acalculous cholecystitis (CAC) has not been documented. History of aortic dissection could be a risk factor of acute and CAC due to relatively decreased splanchnic blood flow. PMID:26232599

  19. Unusual presentation and treatment of isolated spontaneous gastric artery dissection

    PubMed Central

    Lim, Ji Yeon; Choi, Yoon Hee; Lee, Sun Hwa

    2016-01-01

    Epigastric pain is a common symptom in the emergency department. Most epigastric pain is not fatal, except in acute myocardial infarction, aortic dissection, and abdominal aneurysm rupture. Epigastric pain can also be the only symptom of isolated spontaneous gastric artery dissection. We report the case of a 51-year-old woman diagnosed with neurofibromatosis who initially presented with only epigastric pain. She was initially misdiagnosed with gastritis, treated conservatively, and discharged. Two hours later, she returned to the emergency department with persistent epigastric pain and rebound tenderness in the epigastric area. Angiography revealed a left hepatic arterial aneurysm and right gastric artery dissection, which were subsequently embolized. Although extremely rare, patients presenting with epigastric pain at risk of vasculopathies should be considered for isolated spontaneous gastric artery dissection. Early recognition and definitive therapy lead to a favorable outcome. PMID:27752628

  20. Quantitative research.

    PubMed

    Watson, Roger

    2015-04-01

    This article describes the basic tenets of quantitative research. The concepts of dependent and independent variables are addressed and the concept of measurement and its associated issues, such as error, reliability and validity, are explored. Experiments and surveys – the principal research designs in quantitative research – are described and key features explained. The importance of the double-blind randomised controlled trial is emphasised, alongside the importance of longitudinal surveys, as opposed to cross-sectional surveys. Essential features of data storage are covered, with an emphasis on safe, anonymous storage. Finally, the article explores the analysis of quantitative data, considering what may be analysed and the main uses of statistics in analysis.

  1. Multiplex staining of 2-DE gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox specie: Quercus ilex L. subsp. ballota [Desf.] Samp.

    PubMed Central

    Romero-Rodríguez, M. Cristina; Abril, Nieves; Sánchez-Lucas, Rosa; Jorrín-Novo, Jesús V.

    2015-01-01

    As a preliminary step in the phosphoproteome analysis of germinating seeds (0 and 24 h after seed imbibition) and early grown seedlings (216 h after seed imbibition) from a non-orthodox sp. Quercus ilex, a multiplex (SYPRO-Ruby and Pro-Q DPS) staining of high-resolution 2-DE gels was used. By using this protocol it was possible to detect changes in protein-abundance and/or phosphorylation status. This simple approach could be a good complementary alternative to the enrichment protocols used in the search for phosphoprotein candidates. While 482 spots were visualized with SYPRO-Ruby, 222 were with Pro-Q DPS. Statistically significant differences in spot intensity were observed among samples, these corresponding to 85 SYPRO-Ruby-, 20 Pro-Q-DPS-, and 35 SYPRO-Ruby and Pro-Q-DPS-stained spots. Fifty-five phosphoprotein candidates showing qualitative or quantitative differences between samples were subjected to MALDI-TOF-TOF MS analysis, with 20 of them being identified. Identified proteins belonged to five different functional categories, namely: carbohydrate and amino acid metabolism, defense, protein folding, and oxidation-reduction processes. With the exception of a putative cyclase, the other 19 proteins had at least one orthologous phosphoprotein in Arabidopsis thaliana, Medicago truncatula, N. tabacum, and Glycine max. Out of the 20 identified, seven showed differences in intensity in Pro-Q-DPS but not in SYPRO-Ruby-stained gels, including enzymes of the glycolysis and amino acid metabolism. This bears out that theory the regulation of these enzymes occurs at the post-translational level by phosphorylation with no changes at the transcriptional or translational level. This is different from the mechanism reported in orthodox seeds, in which concomitant changes in abundance and phosphorylation status have been observed for these enzymes. PMID:26322061

  2. [Aortic dissection and pregnancy. Apropos of a case].

    PubMed

    Helms, E; Uguen, T; Amaranto, P; Carton, M J; Ducreux, J C; Tempelhoff, C

    1995-03-01

    Aortic dissection is a serious disease which rarely affects young women. In this context, it occurs in nearly one out of two cases during pregnancy, usually during the third term. The authors report acute dissection of the ascending aorta (de Bakey type 2) during pregnancy for which rapid cardiothoracic surgical management as a semi-emergency resulted in a favourable outcome for mother and child.

  3. [Aortic dissection and pregnancy. Apropos of a case].

    PubMed

    Helms, E; Uguen, T; Amaranto, P; Carton, M J; Ducreux, J C; Tempelhoff, C

    1995-03-01

    Aortic dissection is a serious disease which rarely affects young women. In this context, it occurs in nearly one out of two cases during pregnancy, usually during the third term. The authors report acute dissection of the ascending aorta (de Bakey type 2) during pregnancy for which rapid cardiothoracic surgical management as a semi-emergency resulted in a favourable outcome for mother and child. PMID:7487294

  4. Risk of marginal mandibular nerve injury in neck dissection.

    PubMed

    Møller, Martin Nue; Sørensen, Christian Hjort

    2012-02-01

    The immediate and permanent frequency of injury to the marginal mandibular branch of the facial nerve (MMN) after neck dissection has only scarcely been addressed in the medical literature. We investigated the risk of injury in 159 consecutive patients after neck dissection for various reasons in level I B and level II A, respectively. In 95 patients with oral cancer 13 (14%) of the cases had malfunction of the lower lip domain 2 weeks after neck dissection in level I B indicating paresis to the MMN. Follow-up analyses 1-2 years after the operation showed permanent paralysis in 4 to 7% of the cases in whom two of them had the nerve sacrificed for oncologic reasons during the operation. In 18 patients with parotic cancer the corresponding permanent frequency of MMN paralysis was 11.1%. In 46 patients with neck dissection in level II A but not in level I B, no paresis of the MMN was registered. Recognition of the MMN during the operation, pre- or postoperative radiation therapy, re-operation for deep hemorrhage, age, gender or postoperative infection did not have any statistically significant influence on the frequency of MMN injury. In conclusion we found a moderate risk of injury to the MMN after neck dissection in level I B whereas the corresponding risk after level II A dissection was negligible. PMID:21553271

  5. Dynamic Phosphoproteome Analysis of Seedling Leaves in Brachypodium distachyon L. Reveals Central Phosphorylated Proteins Involved in the Drought Stress Response

    PubMed Central

    Yuan, Lin-Lin; Zhang, Ming; Yan, Xing; Bian, Yan-Wei; Zhen, Shou-Min; Yan, Yue-Ming

    2016-01-01

    Drought stress is a major abiotic stress affecting plant growth and development. In this study, we performed the first dynamic phosphoproteome analysis of Brachypodium distachyon L. seedling leaves under drought stress for different times. A total of 4924 phosphopeptides, contained 6362 phosphosites belonging to 2748 phosphoproteins. Rigorous standards were imposed to screen 484 phosphorylation sites, representing 442 unique phosphoproteins. Comparative analyses revealed significant changes in phosphorylation levels at 0, 6, and 24 h under drought stress. The most phosphorylated proteins and the highest phosphorylation level occurred at 6 h. Venn analysis showed that the up-regulated phosphopeptides at 6 h were almost two-fold those at 24 h. Motif-X analysis identified the six motifs: [sP], [Rxxs], [LxRxxs], [sxD], [sF], and [TP], among which [LxRxxs] was also previously identified in B. distachyon. Results from molecular function and protein-protein interaction analyses suggested that phosphoproteins mainly participate in signal transduction, gene expression, drought response and defense, photosynthesis and energy metabolism, and material transmembrane transport. These phosphoproteins, which showed significant changes in phosphorylation levels, play important roles in signal transduction and material transmembrane transport in response to drought conditions. Our results provide new insights into the molecular mechanism of this plant’s abiotic stress response through phosphorylation modification. PMID:27748408

  6. The Tec Kinase-Regulated Phosphoproteome Reveals a Mechanism for the Regulation of Inhibitory Signals in Murine Macrophages.

    PubMed

    Tampella, Giacomo; Kerns, Hannah M; Niu, Deqiang; Singh, Swati; Khim, Socheath; Bosch, Katherine A; Garrett, Meghan E; Moguche, Albanus; Evans, Erica; Browning, Beth; Jahan, Tahmina A; Nacht, Mariana; Wolf-Yadlin, Alejandro; Plebani, Alessandro; Hamerman, Jessica A; Rawlings, David J; James, Richard G

    2015-07-01

    Previous work has shown conflicting roles for Tec family kinases in regulation of TLR-dependent signaling in myeloid cells. In the present study, we performed a detailed investigation of the role of the Tec kinases Btk and Tec kinases in regulating TLR signaling in several types of primary murine macrophages. We demonstrate that primary resident peritoneal macrophages deficient for Btk and Tec secrete less proinflammatory cytokines in response to TLR stimulation than do wild-type cells. In contrast, we found that bone marrow-derived and thioglycollate-elicited peritoneal macrophages deficient for Btk and Tec secrete more proinflammatory cytokines than do wild-type cells. We then compared the phosphoproteome regulated by Tec kinases and LPS in primary peritoneal and bone marrow-derived macrophages. From this analysis we determined that Tec kinases regulate different signaling programs in these cell types. In additional studies using bone marrow-derived macrophages, we found that Tec and Btk promote phosphorylation events necessary for immunoreceptor-mediated inhibition of TLR signaling. Taken together, our results are consistent with a model where Tec kinases (Btk, Tec, Bmx) are required for TLR-dependent signaling in many types of myeloid cells. However, our data also support a cell type-specific TLR inhibitory role for Btk and Tec that is mediated by immunoreceptor activation and signaling via PI3K. PMID:26026062

  7. The Tec kinase-regulated phosphoproteome reveals a mechanism for the regulation of inhibitory signals in murine macrophages

    PubMed Central

    Tampella, Giacomo; Kerns, Hannah M.; Niu, Deqiang; Singh, Swati; Khim, Socheath; Bosch, Katherine A.; Garrett, Meghan E.; Moguche, Albanus; Evans, Erica; Browning, Beth; Jahan, Tahmina A.; Nacht, Mariana; Wolf-Yadlin, Alejandro; Plebani, Alessandro; Hamerman, Jessica A.; Rawlings, David J.; James, Richard G.

    2015-01-01

    Previous work has shown conflicting roles for Tec family kinases in regulation of Toll-like receptor (TLR)-dependent signalling in myeloid cells. In the present study, we performed a detailed investigation of the role of Btk and Tec kinases in regulating TLR signalling in several types of primary murine macrophages. We demonstrate that primary resident peritoneal macrophages deficient for Btk and Tec secrete less pro-inflammatory cytokines in response to TLR stimulation than wild type cells. In contrast, we found that bone marrow-derived and thioglycollate-elicited peritoneal macrophages deficient for Btk and Tec secrete more pro-inflammatory cytokines than wild type cells. We then compared the phosphoproteome regulated by Tec kinases and lipopolysaccharide in primary peritoneal and bone marrow derived macrophages. From this analysis we determined that Tec kinases regulate different signalling programs in these cell types. In additional studies using bone marrow-derived macrophages, we find that Tec and Btk promote phosphorylation events necessary for immunoreceptor-mediated inhibition of TLR signalling. Taken together, our results are consistent with a model where Tec kinases (Btk, Tec, Bmx) are required for TLR-dependent signalling in many types of myeloid cells. However, our data also support a cell type-specific TLR-inhibitory role for Btk and Tec that is mediated by immunoreceptor activation and signalling via PI3K. PMID:26026062

  8. Proteomic and phosphoproteomic analysis of renal cortex in a salt-load rat model of advanced kidney damage

    PubMed Central

    Jiang, Shaoling; He, Hanchang; Tan, Lishan; Wang, Liangliang; Su, Zhengxiu; Liu, Yufeng; Zhu, Hongguo; Zhang, Menghuan; Hou, Fan Fan; Li, Aiqing

    2016-01-01

    Salt plays an essential role in the progression of chronic kidney disease and hypertension. However, the mechanisms underlying pathogenesis of salt-induced kidney damage remain largely unknown. Here, Sprague-Dawley rats, that underwent 5/6 nephrectomy (5/6Nx, a model of advanced kidney damage) or sham operation, were treated for 2 weeks with a normal or high-salt diet. We employed aTiO2 enrichment, iTRAQ labeling and liquid-chromatography tandem mass spectrometry strategy for proteomic and phosphoproteomic profiling of the renal cortex. We found 318 proteins differentially expressed in 5/6Nx group relative to sham group, and 310 proteins significantly changed in response to salt load in 5/6Nx animals. Totally, 1810 unique phosphopeptides corresponding to 550 phosphoproteins were identified. We identified 113 upregulated and 84 downregulated phosphopeptides in 5/6Nx animals relative to sham animals. Salt load induced 78 upregulated and 91 downregulated phosphopeptides in 5/6Nx rats. The differentially expressed phospholproteins are important transporters, structural molecules, and receptors. Protein-protein interaction analysis revealed that the differentially phosphorylated proteins in 5/6Nx group, Polr2a, Srrm1, Gsta2 and Pxn were the most linked. Salt-induced differential phosphoproteins, Myh6, Lmna and Des were the most linked. Altered phosphorylation levels of lamin A and phospholamban were validated. This study will provide new insight into pathogenetic mechanisms of chronic kidney disease and salt sensitivity. PMID:27775022

  9. Global Phosphoproteomic Analysis of Human Skeletal Muscle Reveals a Network of Exercise-Regulated Kinases and AMPK Substrates.

    PubMed

    Hoffman, Nolan J; Parker, Benjamin L; Chaudhuri, Rima; Fisher-Wellman, Kelsey H; Kleinert, Maximilian; Humphrey, Sean J; Yang, Pengyi; Holliday, Mira; Trefely, Sophie; Fazakerley, Daniel J; Stöckli, Jacqueline; Burchfield, James G; Jensen, Thomas E; Jothi, Raja; Kiens, Bente; Wojtaszewski, Jørgen F P; Richter, Erik A; James, David E

    2015-11-01

    Exercise is essential in regulating energy metabolism and whole-body insulin sensitivity. To explore the exercise signaling network, we undertook a global analysis of protein phosphorylation in human skeletal muscle biopsies from untrained healthy males before and after a single high-intensity exercise bout, revealing 1,004 unique exercise-regulated phosphosites on 562 proteins. These included substrates of known exercise-regulated kinases (AMPK, PKA, CaMK, MAPK, mTOR), yet the majority of kinases and substrate phosphosites have not previously been implicated in exercise signaling. Given the importance of AMPK in exercise-regulated metabolism, we performed a targeted in vitro AMPK screen and employed machine learning to predict exercise-regulated AMPK substrates. We validated eight predicted AMPK substrates, including AKAP1, using targeted phosphoproteomics. Functional characterization revealed an undescribed role for AMPK-dependent phosphorylation of AKAP1 in mitochondrial respiration. These data expose the unexplored complexity of acute exercise signaling and provide insights into the role of AMPK in mitochondrial biochemistry.

  10. Global Phosphoproteomic Analysis Reveals the Involvement of Phosphorylation in Aflatoxins Biosynthesis in the Pathogenic Fungus Aspergillus flavus

    PubMed Central

    Ren, Silin; Yang, Mingkun; Li, Yu; Zhang, Feng; Chen, Zhuo; Zhang, Jia; Yang, Guang; Yue, Yuewei; Li, Siting; Ge, Feng; Wang, Shihua

    2016-01-01

    Aspergillus flavus is a pathogenic fungus that produces toxic and carcinogenic aflatoxins and is the causative agent of aflatoxicosis. A growing body of evidence indicates that reversible phosphorylation plays important roles in regulating diverse functions in this pathogen. However, only a few phosphoproteins of this fungus have been identified, which hampers our understanding of the roles of phosphorylation in A. flavus. So we performed a global and site-specific phosphoproteomic analysis of A. flavus. A total of 598 high-confidence phosphorylation sites were identified in 283 phosphoproteins. The identified phosphoproteins were involved in various biological processes, including signal transduction and aflatoxins biosynthesis. Five identified phosphoproteins associated with MAPK signal transduction and aflatoxins biosynthesis were validated by immunoblotting using phospho-specific antibodies. Further functional studies revealed that phosphorylation of the MAP kinase kinase kinase Ste11 affected aflatoxins biosynthesis in A. flavus. Our data represent the results of the first global survey of protein phosphorylation in A. flavus and reveal previously unappreciated roles for phosphorylation in the regulation of aflatoxins production. The generated dataset can serve as an important resource for the functional analysis of protein phosphorylation in A. flavus and facilitate the elucidation of phosphorylated signaling networks in this pathogen. PMID:27667718

  11. Nuclear Phosphoproteomic Screen Uncovers ACLY as Mediator of IL-2-induced Proliferation of CD4+ T lymphocytes.

    PubMed

    Osinalde, Nerea; Mitxelena, Jone; Sánchez-Quiles, Virginia; Akimov, Vyacheslav; Aloria, Kerman; Arizmendi, Jesus M; Zubiaga, Ana M; Blagoev, Blagoy; Kratchmarova, Irina

    2016-06-01

    Anti-cancer immunotherapies commonly rely on the use of interleukin-2 (IL-2) to promote the expansion of T lymphocytes. IL-2- dependent proliferation is the culmination of a complex network of phosphorylation-driven signaling events that impact on gene transcription through mechanisms that are not clearly understood. To study the role of IL-2 in the regulation of nuclear protein function we have performed an unbiased mass spectrometry-based study of the nuclear phosphoproteome of resting and IL-2-treated CD4(+) T lymphocytes. We detected 8521distinct phosphosites including many that are not yet reported in curated phosphorylation databases. Although most phosphorylation sites remained unaffected upon IL-2 treatment, 391 sites corresponding to 288 gene products showed robust IL-2-dependent regulation. Importantly, we show that ATP-citrate lyase (ACLY) is a key phosphoprotein effector of IL-2-mediated T-cell responses. ACLY becomes phosphorylated on serine 455 in T lymphocytes upon IL-2-driven activation of AKT, and depletion or inactivation of ACLY compromises IL-2-promoted T-cell growth. Mechanistically, we demonstrate that ACLY is required for enhancing histone acetylation levels and inducing the expression of cell cycle regulating genes in response to IL-2. Thus, the metabolic enzyme ACLY emerges as a bridge between cytokine signaling and proliferation of T lymphocytes, and may be an attractive candidate target for the development of more efficient anti-cancer immunotherapies.

  12. Phosphoproteomics Reveals Resveratrol-Dependent Inhibition of Akt/mTORC1/S6K1 Signaling

    PubMed Central

    2015-01-01

    Resveratrol, a plant-derived polyphenol, regulates many cellular processes, including cell proliferation, aging and autophagy. However, the molecular mechanisms of resveratrol action in cells are not completely understood. Intriguingly, resveratrol treatment of cells growing in nutrient-rich conditions induces autophagy, while acute resveratrol treatment of cells in a serum-deprived state inhibits autophagy. In this study, we performed a phosphoproteomic analysis after applying resveratrol to serum-starved cells with the goal of identifying the acute signaling events initiated by resveratrol in a serum-deprived state. We determined that resveratrol in serum-starved conditions reduces the phosphorylation of several proteins belonging to the mTORC1 signaling pathway, most significantly, PRAS40 at T246 and S183. Under these same conditions, we also found that resveratrol altered the phosphorylation of several proteins involved in various biological processes, most notably transcriptional modulators, represented by p53, FOXA1, and AATF. Together these data provide a more comprehensive view of both the spectrum of phosphoproteins upon which resveratrol acts as well as the potential mechanisms by which it inhibits autophagy in serum-deprived cells. PMID:25311616

  13. Phosphoproteomics reveals resveratrol-dependent inhibition of Akt/mTORC1/S6K1 signaling.

    PubMed

    Alayev, Anya; Doubleday, Peter F; Berger, Sara Malka; Ballif, Bryan A; Holz, Marina K

    2014-12-01

    Resveratrol, a plant-derived polyphenol, regulates many cellular processes, including cell proliferation, aging and autophagy. However, the molecular mechanisms of resveratrol action in cells are not completely understood. Intriguingly, resveratrol treatment of cells growing in nutrient-rich conditions induces autophagy, while acute resveratrol treatment of cells in a serum-deprived state inhibits autophagy. In this study, we performed a phosphoproteomic analysis after applying resveratrol to serum-starved cells with the goal of identifying the acute signaling events initiated by resveratrol in a serum-deprived state. We determined that resveratrol in serum-starved conditions reduces the phosphorylation of several proteins belonging to the mTORC1 signaling pathway, most significantly, PRAS40 at T246 and S183. Under these same conditions, we also found that resveratrol altered the phosphorylation of several proteins involved in various biological processes, most notably transcriptional modulators, represented by p53, FOXA1, and AATF. Together these data provide a more comprehensive view of both the spectrum of phosphoproteins upon which resveratrol acts as well as the potential mechanisms by which it inhibits autophagy in serum-deprived cells.

  14. Phosphoproteomic analysis of Methanohalophilus portucalensis FDF1T identified the role of protein phosphorylation in methanogenesis and osmoregulation

    PubMed Central

    Wu, Wan-Ling; Lai, Shu-Jung; Yang, Jhih-Tian; Chern, Jeffy; Liang, Suh-Yuen; Chou, Chi-Chi; Kuo, Chih-Horng; Lai, Mei-Chin; Wu, Shih-Hsiung

    2016-01-01

    Methanogens have gained much attention for their metabolic product, methane, which could be an energy substitute but also contributes to the greenhouse effect. One factor that controls methane emission, reversible protein phosphorylation, is a crucial signaling switch, and phosphoproteomics has become a powerful tool for large-scale surveying. Here, we conducted the first phosphorylation-mediated regulation study in halophilic Methanohalophilus portucalensis FDF1T, a model strain for studying stress response mechanisms in osmoadaptation. A shotgun approach and MS-based analysis identified 149 unique phosphoproteins. Among them, 26% participated in methanogenesis and osmolytes biosynthesis pathways. Of note, we uncovered that protein phosphorylation might be a crucial factor to modulate the pyrrolysine (Pyl) incorporation and Pyl-mediated methylotrophic methanogenesis. Furthermore, heterologous expression of glycine sarcosine N-methyltransferase (GSMT) mutant derivatives in the osmosensitive Escherichia coli MKH13 revealed that the nonphosphorylated T68A mutant resulted in increased salt tolerance. In contrast, mimic phosphorylated mutant T68D proved defective in both enzymatic activity and salinity tolerance for growth. Our study provides new insights into phosphorylation modification as a crucial role of both methanogenesis and osmoadaptation in methanoarchaea, promoting biogas production or reducing future methane emission in response to global warming and climate change. PMID:27357474

  15. Phosphoproteomic analysis of Methanohalophilus portucalensis FDF1(T) identified the role of protein phosphorylation in methanogenesis and osmoregulation.

    PubMed

    Wu, Wan-Ling; Lai, Shu-Jung; Yang, Jhih-Tian; Chern, Jeffy; Liang, Suh-Yuen; Chou, Chi-Chi; Kuo, Chih-Horng; Lai, Mei-Chin; Wu, Shih-Hsiung

    2016-01-01

    Methanogens have gained much attention for their metabolic product, methane, which could be an energy substitute but also contributes to the greenhouse effect. One factor that controls methane emission, reversible protein phosphorylation, is a crucial signaling switch, and phosphoproteomics has become a powerful tool for large-scale surveying. Here, we conducted the first phosphorylation-mediated regulation study in halophilic Methanohalophilus portucalensis FDF1(T), a model strain for studying stress response mechanisms in osmoadaptation. A shotgun approach and MS-based analysis identified 149 unique phosphoproteins. Among them, 26% participated in methanogenesis and osmolytes biosynthesis pathways. Of note, we uncovered that protein phosphorylation might be a crucial factor to modulate the pyrrolysine (Pyl) incorporation and Pyl-mediated methylotrophic methanogenesis. Furthermore, heterologous expression of glycine sarcosine N-methyltransferase (GSMT) mutant derivatives in the osmosensitive Escherichia coli MKH13 revealed that the nonphosphorylated T68A mutant resulted in increased salt tolerance. In contrast, mimic phosphorylated mutant T68D proved defective in both enzymatic activity and salinity tolerance for growth. Our study provides new insights into phosphorylation modification as a crucial role of both methanogenesis and osmoadaptation in methanoarchaea, promoting biogas production or reducing future methane emission in response to global warming and climate change. PMID:27357474

  16. Quantitative Thinking.

    ERIC Educational Resources Information Center

    DuBridge, Lee A.

    An appeal for more research to determine how to educate children as effectively as possible is made. Mathematics teachers can readily examine the educational problems of today in their classrooms since learning progress in mathematics can easily be measured and evaluated. Since mathematics teachers have learned to think in quantitative terms and…

  17. QUANTITATIVE MORPHOLOGY

    EPA Science Inventory

    Abstract: In toxicology, the role of quantitative assessment of brain morphology can be understood in the context of two types of treatment-related alterations. One type of alteration is specifically associated with treatment and is not observed in control animals. Measurement ...

  18. Ultrasonic Dissection versus Conventional Dissection for Pancreatic Surgery: A Meta-Analysis

    PubMed Central

    2016-01-01

    Background. The role of ultrasonic dissection (UD) in pancreatic surgery remains controversial. The aim of this meta-analysis was to evaluate the clinical effect of UD in pancreatic surgery when compared with conventional dissection (CD). Materials and Methods. A comprehensive literature search was performed to identify eligible studies that compared UD with CD for pancreatic surgery in PubMed, EMBASE, Web of Science, and the Cochrane Library. Risk ratio (RR) or mean difference with 95% confidence interval (CI) was calculated. Results. Six studies were included with a total of 215 patients undergoing UD and 210 undergoing CD. In comparison with CD in distal pancreatectomy, UD was associated with lower rates of pancreatic fistula (RR = 0.46, 95% CI: 0.27–0.76) and abdominal abscess and shorter operation time and hospital stay (P < 0.05). In pancreaticoduodenectomy, there was no significant difference in pancreatic fistula rate between two groups (RR = 0.79, 95% CI: 0.48–1.29). However, the significantly less intraoperative blood loss and the transfused blood unit were found in patients receiving UD (P < 0.05). Conclusions. The results of this meta-analysis show that, in comparison with CD, UD is associated with better perioperative outcomes in pancreatic surgery. PMID:26880891

  19. Applications of quantitative whole body autoradiographic technique in radiopharmaceutical research

    SciTech Connect

    Som, P.; Oster, Z.H.; Yonekura, Y.; Meyer, M.A.; Fand, I.; Brill, A.B.

    1982-01-01

    The routine evaluation of radiopharmaceuticals involves dissecting tissue distribution studies (DTDS) and gamma or positron imaging. DTDS have the following disadvantages: since not all tissues can always be sampled, sites of radiopharmaceutical uptake may be missed and because the procedure involves weighing of dissected tissue samples, the spatial resolution of this method is low and determined by the smallest amount that can be weighed accurately. Gamma camera imaging and positron emission tomography though more comprehensive in evaluating the global distribution of a compound, have relative low spatial resolution. Whole body autoradiography of small animals has a much higher spatial resolution as compared to the above and depicts the global distribution of radiopharmaceuticals. A computer-assisted quantification method of WBARG applied to positron, beta, and gamma emitters will complement the method by producing quantitative values comparable to those obtained by dissection and direct tissue counting, with the advantages of depicting the global distribution at high spatial resolution.

  20. Coronary artery dissection with rupture of aortic valve commissure following type A aortic dissection: the role of 64-slice MDCT.

    PubMed

    Das, K M; Abdou, Sayed M; El-Menyar, Ayman; Ayman, El Menyar; Khulaifi, A A; Nabti, A L

    2008-01-01

    A rare case of bilateral coronary artery dissection with rupture of aortic valve commissure following type A aortic dissection is described. 64-slice multidetector computed tomography (MDCT) was able to demonstrate both this findings along with involvement of other neck vessels. TEE demonstrated the severity and mechanisms of aortic valve damage and assisted the surgeon in valve repair. MDCT has played an invaluable role in the diagnosis of the abnormal details of such life-threatening vascular complications. PMID:18384568

  1. Treatment of Ruptured Vertebral Artery Dissecting Aneurysms

    PubMed Central

    Hamasaki, Osamu; Ikawa, Fusao; Hidaka, Toshikazu; Kurokawa, Yasuharu; Yonezawa, Ushio

    2014-01-01

    Summary We evaluated the outcomes of endovascular or surgical treatment of ruptured vertebral artery dissecting aneurysms (VADAs), and investigated the relations between treatment complications and the development and location of the posterior inferior cerebellar artery (PICA). We treated 14 patients (12 men, two women; mean age, 56.2 years) with ruptured VADAs between March 1999 and June 2012 at our hospital. Six and eight patients had Hunt and Hess grades 1-3 and 4-5, respectively. Twelve patients underwent internal endovascular trapping, one underwent proximal endovascular occlusion alone, and one underwent proximal endovascular occlusion in the acute stage and occipital artery (OA)-PICA anastomosis and surgical trapping in the chronic stage. The types of VADA based on their location relative to the ipsilateral PICA were distal, PICA-involved, and non-PICA in nine, two, and three patients, respectively. The types of PICA based on their development and location were bilateral anterior inferior cerebellar artery (AICA)-PICA, ipsilateral AICA-PICA, extradural, and intradural type in one, two, two, and nine patients, respectively. Two patients with high anatomical risk developed medullary infarction, but their midterm outcomes were better than in previous reports. The modified Rankin scale indicated grades 0-2, 3-5, and 6 in eight, three, and three patients, respectively. A good outcome is often obtained in the treatment of ruptured VADA using internal endovascular trapping, except in the PICA-involved type, even with high-grade subarachnoid hemorrhage. Treatment of the PICA-involved type is controversial. The anatomical location and development of PICA may be predicted by complications with postoperative medullary infarction. PMID:24976093

  2. "And afterwards your body to be given for public dissection": a history of the murderers dissected in Glasgow and the west of Scotland.

    PubMed

    Kennedy, S S; McLeod, K J; McDonald, S W

    2001-02-01

    Between 1752 and 1832, the bodies of hanged murderers were dissected or gibbeted. During this period, 38 murderers were executed in the West of Scotland. The bodies of at least 23 were dissected in Glasgow. The stories of these murders are recounted. Insight is also given into the attitudes of the public and the anatomists to dissection of executed murderers.

  3. A comparison of retention of anatomical knowledge in an introductory college biology course: Traditional dissection vs. virtual dissection

    NASA Astrophysics Data System (ADS)

    Taeger, Kelli Rae

    Dissection has always played a crucial role in biology and anatomy courses at all levels of education. However, in recent years, ethical concerns, as well as improved technology, have brought to the forefront the issue of whether virtual dissection is as effective or whether it is more effective than traditional dissection. Most prior research indicated the two methods produced equal results. However, none of those studies examined retention of information past the initial test of knowledge. Two groups of college students currently enrolled in an introductory level college biology course were given one hour to complete a frog dissection. One group performed a traditional frog dissection, making cuts in an actual preserved frog specimen with scalpels and scissors. The other group performed a virtual frog dissection, using "The Digital Frog 2" software. Immediately after the dissections were completed, each group was given an examination consisting of questions on actual specimens, pictures generated from the computer software, and illustrations that neither group had seen. Two weeks later, unannounced, the groups took the same exam in order to test retention. The traditional dissection group scored significantly higher on two of the three sections, as well as the total score on the initial exam. However, with the exception of specimen questions (on which the traditional group retained significantly more information), there was no significant difference in the retention from exam 1 to exam 2 between the two groups. These results, along with the majority of prior studies, show that the two methods produce, for the most part, the same end results. Therefore, the decision of which method to employ should be based on the goals and preferences of the instructor(s) and the department. If that department's goals include: Being at the forefront of new technology, increasing time management, increasing student: teacher ratio for economic reasons, and/or ethical issues, then

  4. Treatment of ruptured intracranial dissecting aneurysms in Hong Kong

    PubMed Central

    Wong, George Kwok Chu; Tang, Hoi Bun; Poon, Wai Sang; Yu, Simon Chun Ho

    2010-01-01

    Background: Data suggests that hemorrhagic presentations occur in 20% of internal carotid artery dissections and 50% of vertebral artery dissections. A Finnish study has reported favorable outcomes in only 32% of patients. We aimed to review the epidemiology and management outcomes in a Chinese population. Methods: We reviewed the aneurysmal subarachnoid hemorrhage registry of patients who presented with intracranial dissecting aneurysms at a neurosurgical center in Hong Kong over a five-year period. Results: A total of 23 patients with intracranial dissecting aneurysms were identified, accounting for 8% of all spontaneous subarachnoid hemorrhage patients. Forty-eight percent of the patients identified were treated by main trunk occlusion and 39% were treated by embolization or stent-assisted embolization or stent alone. Thirteen percent were managed by craniotomy and trapping or wrapping. Favorable outcomes at six months were achieved in 67%. Conclusions: Patients with intracranial dissecting aneurysms account for a significant proportion of the cases of spontaneous subarachnoid hemorrhage in our population. Carefully selected endovascular and microsurgical treatments can lead to management outcomes similar to patients with saccular aneurysms. PMID:21206536

  5. Traumatic internal carotid artery dissections caused by blunt softball injuries.

    PubMed

    Schievink, W I; Atkinson, J L; Bartleson, J D; Whisnant, J P

    1998-03-01

    This report describes recently treated patients with carotid artery dissection caused by blunt softball injuries, as well as the results of a study of carotid artery trauma in a community. Data obtained through the medical records linkage system used for epidemiologic studies in Olmsted County, MN were used to identify all cases of traumatic internal carotid artery dissection diagnosed from 1987 through 1994. Four patients with traumatic internal carotid artery dissections were identified during the 8-year period under study. In two patients (50%) the carotid dissection was a result of the direct impact of a softball. A 39-year-old-man, who developed transient cerebral ischemic symptoms, and a 35-year-old woman, who developed a painful Horner's syndrome, were struck by a softball on the anterolateral aspect of the neck. Both patients had a low carotid bifurcation. These data suggest that internal carotid artery dissections may be underrecognized sequelae of direct softball injuries to the anterolateral neck. A low carotid bifurcation may be a risk factor for such injuries.

  6. Unraveling pancreatic islet biology by quantitative proteomics

    SciTech Connect

    Zhou, Jianying; Dann, Geoffrey P.; Liew, Chong W.; Smith, Richard D.; Kulkarni, Rohit N.; Qian, Weijun

    2011-08-01

    The pancreatic islets of Langerhans play a critical role in maintaining blood glucose homeostasis by secreting insulin and several other important peptide hormones. Impaired insulin secretion due to islet dysfunction is linked to the pathogenesis underlying both Type 1 and Type 2 diabetes. Over the past 5 years, emerging proteomic technologies have been applied to dissect the signaling pathways that regulate islet functions and gain an understanding of the mechanisms of islet dysfunction relevant to diabetes. Herein, we briefly review some of the recent quantitative proteomic studies involving pancreatic islets geared towards gaining a better understanding of islet biology relevant to metabolic diseases.

  7. Bud set in poplar--genetic dissection of a complex trait in natural and hybrid populations.

    PubMed

    Rohde, Antje; Storme, Véronique; Jorge, Véronique; Gaudet, Muriel; Vitacolonna, Nicola; Fabbrini, Francesco; Ruttink, Tom; Zaina, Giusi; Marron, Nicolas; Dillen, Sophie; Steenackers, Marijke; Sabatti, Maurizio; Morgante, Michele; Boerjan, Wout; Bastien, Catherine

    2011-01-01

    • The seasonal timing of growth events is crucial to tree distribution and conservation. The seasonal growth cycle is strongly adapted to the local climate that is changing because of global warming. We studied bud set as one cornerstone of the seasonal growth cycle in an integrative approach. • Bud set was dissected at the phenotypic level into several components, and phenotypic components with most genetic variation were identified. While phenotypic variation resided in the timing of growth cessation, and even so more in the duration from growth cessation to bud set, the timing of growth cessation had a stronger genetic component in both natural and hybrid populations. • Quantitative trait loci (QTL) were identified for the most discriminative phenotypic bud-set components across four poplar pedigrees. The QTL from different pedigrees were recurrently detected in six regions of the poplar genome. • These regions of 1.83-4.25 Mbp in size, containing between 202 and 394 genes, form the basis for further molecular-genetic dissection of bud set.

  8. Dissecting the Impact of Matrix Anchorage and Elasticity in Cell Adhesion

    PubMed Central

    Pompe, Tilo; Glorius, Stefan; Bischoff, Thomas; Uhlmann, Ina; Kaufmann, Martin; Brenner, Sebastian; Werner, Carsten

    2009-01-01

    Abstract Extracellular matrices determine cellular fate decisions through the regulation of intracellular force and stress. Previous studies suggest that matrix stiffness and ligand anchorage cause distinct signaling effects. We show herein how defined noncovalent anchorage of adhesion ligands to elastic substrates allows for dissection of intracellular adhesion signaling pathways related to matrix stiffness and receptor forces. Quantitative analysis of the mechanical balance in cell adhesion using traction force microscopy revealed distinct scalings of the strain energy imparted by the cells on the substrates dependent either on matrix stiffness or on receptor force. Those scalings suggested the applicability of a linear elastic theoretical framework for the description of cell adhesion in a certain parameter range, which is cell-type-dependent. Besides the deconvolution of biophysical adhesion signaling, site-specific phosphorylation of focal adhesion kinase, dependent either on matrix stiffness or on receptor force, also demonstrated the dissection of biochemical signaling events in our approach. Moreover, the net contractile moment of the adherent cells and their strain energy exerted on the elastic substrate was found to be a robust measure of cell adhesion with a unifying power-law scaling exponent of 1.5 independent of matrix stiffness. PMID:19843448

  9. 3D segmentation of the true and false lumens on CT aortic dissection images

    NASA Astrophysics Data System (ADS)

    Fetnaci, Nawel; Łubniewski, Paweł; Miguel, Bruno; Lohou, Christophe

    2013-03-01

    Our works are related to aortic dissections which are a medical emergency and can quickly lead to death. In this paper, we want to retrieve in CT images the false and the true lumens which are aortic dissection features. Our aim is to provide a 3D view of the lumens that we can difficultly obtain either by volume rendering or by another visualization tool which only directly gives the outer contour of the aorta; or by other segmentation methods because they mainly directly segment either only the outer contour of the aorta or other connected arteries and organs both. In our work, we need to segment the two lumens separately; this segmentation will allow us to: distinguish them automatically, facilitate the landing of the aortic prosthesis, propose a virtual 3d navigation and do quantitative analysis. We chose to segment these data by using a deformable model based on the fast marching method. In the classical fast marching approach, a speed function is used to control the front propagation of a deforming curve. The speed function is only based on the image gradient. In our CT images, due to the low resolution, with the fast marching the front propagates from a lumen to the other; therefore, the gradient data is insufficient to have accurate segmentation results. In the paper, we have adapted the fast marching method more particularly by modifying the speed function and we succeed in segmenting the two lumens separately.

  10. Aortic dissection accompanied by preeclampsia in a postpartum young woman

    PubMed Central

    Park, Jin-Wan; Kim, Su-Mi; Yu, Gyu-Bong

    2016-01-01

    Aortic dissection is very rare in obstetrics, but it is a fatal disease. A 37-weeks primigravida woman with dyspnea and pitting edema presented to our emergency room. The patient was diagnosed with preeclampsia and underwent an emergency cesarean section under spinal anesthesia. The patient complained of severe dyspnea after the cesarean section, and the chest computed tomography scan was done. With the finding of aortic dissection, cardiopulmonary arrest occurred 5 hours after the cesarean section, and the patient died without reaction to cardio-pulmonary resuscitation. If a patient with preeclampsia complains of severe dyspnea or chest pain, aortic dissection needs to be suspected and a diagnosis should not be delayed. PMID:27668205

  11. Digital dissection system for medical school anatomy training

    NASA Astrophysics Data System (ADS)

    Augustine, Kurt E.; Pawlina, Wojciech; Carmichael, Stephen W.; Korinek, Mark J.; Schroeder, Kathryn K.; Segovis, Colin M.; Robb, Richard A.

    2003-05-01

    As technology advances, new and innovative ways of viewing and visualizing the human body are developed. Medicine has benefited greatly from imaging modalities that provide ways for us to visualize anatomy that cannot be seen without invasive procedures. As long as medical procedures include invasive operations, students of anatomy will benefit from the cadaveric dissection experience. Teaching proper technique for dissection of human cadavers is a challenging task for anatomy educators. Traditional methods, which have not changed significantly for centuries, include the use of textbooks and pictures to show students what a particular dissection specimen should look like. The ability to properly carry out such highly visual and interactive procedures is significantly constrained by these methods. The student receives a single view and has no idea how the procedure was carried out. The Department of Anatomy at Mayo Medical School recently built a new, state-of-the-art teaching laboratory, including data ports and power sources above each dissection table. This feature allows students to access the Mayo intranet from a computer mounted on each table. The vision of the Department of Anatomy is to replace all paper-based resources in the laboratory (dissection manuals, anatomic atlases, etc.) with a more dynamic medium that will direct students in dissection and in learning human anatomy. Part of that vision includes the use of interactive 3-D visualization technology. The Biomedical Imaging Resource (BIR) at Mayo Clinic has developed, in collaboration with the Department of Anatomy, a system for the control and capture of high resolution digital photographic sequences which can be used to create 3-D interactive visualizations of specimen dissections. The primary components of the system include a Kodak DC290 digital camera, a motorized controller rig from Kaidan, a PC, and custom software to synchronize and control the components. For each dissection procedure, the

  12. NSAID-induced pyloric stenosis leading to oesophageal intramucosal dissection.

    PubMed

    Tey, Kai Rou; Kemmerly, Thomas; Banerjee, Bhaskar

    2016-01-01

    We describe a rare case of a 75-year-old woman with significant non-steroidal anti-inflammatory drug (NSAID) use who presented with haematemesis. Upper endoscopy revealed a large (9 cm) intramucosal dissection of the oesophagus without extension into the gastro-oesophageal junction and a severely narrowed pylorus. We postulate that she developed pyloric stenosis due to peptic ulcer disease from chronic NSAID use. This then led to gastro-oesophageal reflux. Undigested pills in the refluxate had contacted oesophageal mucosa, leading to pill-induced oesophageal injury. This, along with vomiting, is postulated to have led to the oesophageal intramucosal dissection. She improved with conservative medical management with a clear liquid diet and proton pump inhibitors, and a follow-up upper endoscopy 1 week later showed recovery of the previously seen intramucosal dissection. PMID:27199442

  13. Dissection of the atrial wall after mitral valve replacement.

    PubMed Central

    Lukács, L; Kassai, I; Lengyel, M

    1996-01-01

    We describe an unusual sequela of mitral valve replacement in a 50-year-old woman who had undergone a closed mitral commissurotomy in 1975. She was admitted to our hospital because of mitral restenosis in November 1993, at which time her mitral valve was replaced with a mechanical prosthesis. On the 8th postoperative day, the patient developed symptoms of heart failure; transesophageal echocardiography revealed dissection and rupture of the left atrial wall. At prompt reoperation, we found an interlayer dissection and rupture of the atrial wall into the left atrium. We repaired the ruptured atrial wall with a prosthetic patch. The postoperative course was uneventful, and postoperative transesophageal echocardiography showed normal prosthetic valve function and no dissection. Images PMID:8680278

  14. Spontaneous coronary artery dissection in a patient with bacterial meningitis

    PubMed Central

    Kinoshita, Kensuke; Tsunoda, Yoshiya; Watanabe, Shigeyuki; Tokuda, Yasuharu

    2013-01-01

    A 40-year-old man was admitted to our hospital because of the acute onset of fever and headache, which were attributed to bacterial meningitis. Antibiotic treatment was initiated and his condition gradually improved. On day 5 after admission, immediately after masturbation, he developed abrupt onset of severe chest pain and cold sweat and the ECG suggested acute anterior myocardial infarction. Immediate coronary angiography revealed spontaneous dissection of the left anterior descending artery. After conservative management, his cardiac function improved. Acute coronary syndrome may be rarely caused by spontaneous coronary artery dissection. Sepsis was considered as a probable trigger for spontaneous coronary artery dissection, possibly through vascular damage from increased nitric oxide and sympathetic nervous over-activation. PMID:24194165

  15. Traumatic Carotid Artery Dissection: A Different Entity without Specific Guidelines

    PubMed Central

    Galyfos, George; Filis, Konstantinos; Sigala, Fragiska; Sianou, Argiri

    2016-01-01

    According to literature data, there are no distinct guidelines regarding the proper diagnostic and therapeutic management of traumatic carotid artery dissection (TCAD). Although most of cases evaluated in research studies refer to spontaneous carotid artery dissection, traumatic cases demand special considerations as far as diagnosis and treatment are concerned. Although both types of dissection share some common characteristics, a patient with TCAD usually presents with several concomitant injuries as well as a higher bleeding risk, thus complicating decision making in such patients. Therefore, aim of this review is to present available data regarding epidemiology, clinical presentation, diagnostics and treatment strategy in cases with TCAD in order to produce useful conclusions for everyday clinical practice. PMID:27051653

  16. Postmortem Aortic Dissection: An Artifact of the Embalming Process.

    PubMed

    Rae, Guenevere; Husain, Mujtaba; McGoey, Robin; Swartz, William

    2016-01-01

    Aortic dissection (AD) is a serious condition that affects 3/100,000 individuals a year. Recently, a case report was published describing an embalmed patient with an aortic dissection. The purpose of this study was to examine the frequency of AD among 80 embalmed cadavers and confirm the AD with histopathologic evaluation. In seven cases of grossly identified AD, six were determined to be due to the embalming procedure and only one case of true antemortem AD was confirmed. These results suggest that aortic morphology can be altered by administration of the embalming fluid and that alterations can mimic AD, not only on gross inspection but also on postmortem imaging. Awareness of this embalming artifact may prevent misdiagnosis of an aortic dissection in an embalmed patient, a point particularly useful for autopsy pathology that may include postmortem examination of an embalmed patient.

  17. Bilateral chylothorax following neck dissection: a case report

    PubMed Central

    2014-01-01

    Background Chylothorax is an extremely rare but potentially life-threatening complication after radical neck dissection. We report the case of a bilateral chylothorax after total thyroidectomy and cervico-central and cervico-lateral lymphadenectomy for thyroid carcinoma. Case presentation A 40-year-old European woman underwent total thyroidectomy and neck dissection for papillary thyroid carcinoma. Postoperatively she developed dyspnoea and pleural effusion. A chylothorax was found and the initial conservative therapy was not successful. She had to be operated on again and the thoracic duct was legated. Conclusion The case presentation reports a very rare complication after total thyroidectomy and neck dissection, but it has to be kept in mind to prevent dangerous complications. PMID:24885488

  18. Aortic dissection accompanied by preeclampsia in a postpartum young woman.

    PubMed

    Park, Jin-Wan; Kim, Su-Mi; Yu, Gyu-Bong; Kang, Yun-Dan

    2016-09-01

    Aortic dissection is very rare in obstetrics, but it is a fatal disease. A 37-weeks primigravida woman with dyspnea and pitting edema presented to our emergency room. The patient was diagnosed with preeclampsia and underwent an emergency cesarean section under spinal anesthesia. The patient complained of severe dyspnea after the cesarean section, and the chest computed tomography scan was done. With the finding of aortic dissection, cardiopulmonary arrest occurred 5 hours after the cesarean section, and the patient died without reaction to cardio-pulmonary resuscitation. If a patient with preeclampsia complains of severe dyspnea or chest pain, aortic dissection needs to be suspected and a diagnosis should not be delayed. PMID:27668205

  19. Free manual of cadaver dissection modifiable by other anatomists.

    PubMed

    Chung, Beom Sun; Chung, Min Suk

    2015-06-01

    Even in the rapidly changing field of cadaver dissection, published guide books still play an important role in the anatomy lab. However, commercial manuals with lengthy volumes and inflexible copyrights have several limitations which can be complemented by open-source manuals. Recently, the authors have manufactured and distributed a free electronic dissection manual (anatomy.co.kr), where descriptions are written concisely and images are drawn schematically. Moreover, simplified signs are employed to represent the cadaver viewing angles and manner of dissection. Based on the original files of this manual, other anatomists can revise and utilize the descriptions and figures. We expect many updated versions of our manual to be shared between students all over the world.

  20. Are all hands-on activities equally effective? Effect of using plastic models, organ dissections, and virtual dissections on student learning and perceptions.

    PubMed

    Lombardi, Sara A; Hicks, Reimi E; Thompson, Katerina V; Marbach-Ad, Gili

    2014-03-01

    This study investigated the impact of three commonly used cardiovascular model-assisted activities on student learning and student attitudes and perspectives about science. College students enrolled in a Human Anatomy and Physiology course were randomly assigned to one of three experimental groups (organ dissections, virtual dissections, or plastic models). Each group received a 15-min lecture followed by a 45-min activity with one of the treatments. Immediately after the lesson and then 2 mo later, students were tested on anatomy and physiology knowledge and completed an attitude survey. Students who used plastic models achieved significantly higher overall scores on both the initial and followup exams than students who performed organ or virtual dissections. On the initial exam, students in the plastic model and organ dissection treatments scored higher on anatomy questions than students who performed virtual dissections. Students in the plastic model group scored higher than students who performed organ dissections on physiology questions. On the followup exam, when asked anatomy questions, students in the plastic model group scored higher than dissection students and virtual dissection students. On attitude surveys, organ dissections had higher perceived value and were requested for inclusion in curricula twice as often as any other activity. Students who performed organ dissections were more likely than the other treatment groups to agree with the statement that "science is fun," suggesting that organ dissections may promote positive attitudes toward science. The findings of this study provide evidence for the importance of multiple types of hands-on activities in anatomy laboratory courses.

  1. Phosphoproteomic Analysis of KSHV-Infected Cells Reveals Roles of ORF45-Activated RSK during Lytic Replication

    PubMed Central

    Avey, Denis; Tepper, Sarah; Li, Wenwei; Turpin, Zachary; Zhu, Fanxiu

    2015-01-01

    Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) is an oncogenic virus which has adapted unique mechanisms to modulate the cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling pathways, including the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. We have previously shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK, a major functional mediator of ERK/MAPK signaling) during KSHV lytic replication. ORF45-activated RSK is required for optimal KSHV lytic gene expression and progeny virion production, though the underlying mechanisms downstream of this activation are still unclear. We hypothesized that the activation of RSK by ORF45 causes differential phosphorylation of cellular and viral substrates, affecting biological processes essential for efficient KSHV lytic replication. Accordingly, we observed widespread and significant differences in protein phosphorylation upon induction of lytic replication. Mass-spectrometry-based phosphoproteomic screening identified putative substrates of ORF45-activated RSK in KSHV-infected cells. Bioinformatic analyses revealed that nuclear proteins, including several transcriptional regulators, were overrepresented among these candidates. We validated the ORF45/RSK-dependent phosphorylation of several putative substrates by employing KSHV BAC mutagenesis, kinase inhibitor treatments, and/or CRISPR-mediated knockout of RSK in KSHV-infected cells. Furthermore, we assessed the consequences of knocking out these substrates on ORF45/RSK-dependent regulation of gene expression and KSHV progeny virion production. Finally, we show data to support that ORF45 regulates the translational efficiency of a subset of viral/cellular genes with complex secondary structure in their 5’ UTR. Altogether, these data shed light on the mechanisms by which KSHV ORF45 manipulates

  2. Proteome and phosphoproteome analysis of honeybee (Apis mellifera) venom collected from electrical stimulation and manual extraction of the venom gland

    PubMed Central

    2013-01-01

    Background Honeybee venom is a complicated defensive toxin that has a wide range of pharmacologically active compounds. Some of these compounds are useful for human therapeutics. There are two major forms of honeybee venom used in pharmacological applications: manually (or reservoir disrupting) extracted glandular venom (GV), and venom extracted through the use of electrical stimulation (ESV). A proteome comparison of these two venom forms and an understanding of the phosphorylation status of ESV, are still very limited. Here, the proteomes of GV and ESV were compared using both gel-based and gel-free proteomics approaches and the phosphoproteome of ESV was determined through the use of TiO2 enrichment. Results Of the 43 proteins identified in GV, < 40% were venom toxins, and > 60% of the proteins were non-toxic proteins resulting from contamination by gland tissue damage during extraction and bee death. Of the 17 proteins identified in ESV, 14 proteins (>80%) were venom toxic proteins and most of them were found in higher abundance than in GV. Moreover, two novel proteins (dehydrogenase/reductase SDR family member 11-like and histone H2B.3-like) and three novel phosphorylation sites (icarapin (S43), phospholipase A-2 (T145), and apamin (T23)) were identified. Conclusions Our data demonstrate that venom extracted manually is different from venom extracted using ESV, and these differences may be important in their use as pharmacological agents. ESV may be more efficient than GV as a potential pharmacological source because of its higher venom protein content, production efficiency, and without the need to kill honeybee. The three newly identified phosphorylated venom proteins in ESV may elicit a different immune response through the specific recognition of antigenic determinants. The two novel venom proteins extend our proteome coverage of honeybee venom. PMID:24199871

  3. Dissecting cellular biomechanics with a laser

    NASA Astrophysics Data System (ADS)

    Hutson, M. Shane

    2011-10-01

    The biological tissues of a developing organism are built and reshaped by the mechanical behavior of individual cells. We probe the relevant cellular mechanics in vivo using laser-microsurgery -- both qualitatively, to assess whether removal of specific cells alters the dynamics of tissue reshaping, and quantitatively, to measure sub-cellular mechanical properties and stresses. I will detail two quantitative microsurgical measurements. The first uses a laser to drill a sub-cellular hole in a sheet of cells. The subsequent retraction of surrounding cells allows one to infer the local mechanical stress. The second uses a laser to isolate a single cell from the rest of a cell sheet. Isolation is accomplished on a microsecond time scale by holographically shaping a single laser pulse. The subsequent retraction (or expansion) of the isolated cell allows one to separate and quantify the effects of internal and external stresses in the determination of cell shape. I will discuss application of these techniques to the time-dependent biomechanics of epithelial tissues during early fruit fly embryogenesis -- specifically during the processes of germband retraction and dorsal closure.

  4. The hemostatic disturbance in patients with acute aortic dissection

    PubMed Central

    Guan, Xinliang; Li, Jiachen; Gong, Ming; Lan, Feng; Zhang, Hongjia

    2016-01-01

    Abstract Coagulopathy is still a frequent complication in the surgical treatment of acute aortic dissection. However, the physiopathology of surgically induced coagulopathy has never been systematically and comprehensively studied in patients with acute aortic dissection. The aim of the present study was to describe the perioperative hemostatic system in patients with acute aortic dissection. The 87 patients who underwent aortic arch surgery for acute Stanford type A aortic dissection from January 2013 to September 2015 were enrolled in this study. The perioperative biomarkers of hemostatic system were evaluated using standard laboratory tests and enzyme-linked immunosorbent assays (ELISAs) at 5 time points: anesthesia induction (T1), lowest nasopharyngeal temperature (T2), protamine reversal (T3), 4 hours after surgery (T4), and 24 hours after surgery (T5). The ELISAs biomarkers revealed activation of coagulation (thrombin-antithrombin III complex [TAT] and prothrombin fragment 1 + 2 [F1 + 2] were elevated), suppression of anticoagulation (antithrombin III [AT III] levels were depressed), and activation of fibrinolysis (plasminogen was decreased and plasmin-antiplasmin complex [PAP] was elevated). The standard laboratory tests also demonstrated that surgery resulted in a significant reduction in platelet counts and fibrinogen concentration. Systemic activation of coagulation and fibrinolysis, and inhibition of anticoagulation were observed during the perioperative period in patients with acute aortic dissection. Indeed, these patients exhibited consumption coagulopathy and procoagulant state perioperatively. Therefore, we believe that this remarkable disseminated intravascular coagulation (DIC)-like coagulopathy has a high risk of bleeding and may influence postoperative outcome of patients with acute aortic dissection. PMID:27603366

  5. Integrated phosphoproteomic and metabolomic profiling reveals NPM-ALK-mediated phosphorylation of PKM2 and metabolic reprogramming in anaplastic large cell lymphoma.

    PubMed

    McDonnell, Scott R P; Hwang, Steven R; Rolland, Delphine; Murga-Zamalloa, Carlos; Basrur, Venkatesha; Conlon, Kevin P; Fermin, Damian; Wolfe, Thomas; Raskind, Alexander; Ruan, Chunhai; Jiang, Jian-Kang; Thomas, Craig J; Hogaboam, Cory M; Burant, Charles F; Elenitoba-Johnson, Kojo S J; Lim, Megan S

    2013-08-01

    The mechanisms underlying the pathogenesis of the constitutively active tyrosine kinase nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expressing anaplastic large cell lymphoma are not completely understood. Here we show using an integrated phosphoproteomic and metabolomic strategy that NPM-ALK induces a metabolic shift toward aerobic glycolysis, increased lactate production, and biomass production. The metabolic shift is mediated through the anaplastic lymphoma kinase (ALK) phosphorylation of the tumor-specific isoform of pyruvate kinase (PKM2) at Y105, resulting in decreased enzymatic activity. Small molecule activation of PKM2 or expression of Y105F PKM2 mutant leads to reversal of the metabolic switch with increased oxidative phosphorylation and reduced lactate production coincident with increased cell death, decreased colony formation, and reduced tumor growth in an in vivo xenograft model. This study provides comprehensive profiling of the phosphoproteomic and metabolomic consequences of NPM-ALK expression and reveals a novel role of ALK in the regulation of multiple components of cellular metabolism. Our studies show that PKM2 is a novel substrate of ALK and plays a critical role in mediating the metabolic shift toward biomass production and tumorigenesis.

  6. Dissection of brassinosteroid-regulated proteins in rice embryos during germination by quantitative proteomics

    PubMed Central

    Li, Qian-Feng; Xiong, Min; Xu, Peng; Huang, Li-Chun; Zhang, Chang-Quan; Liu, Qiao-Quan

    2016-01-01

    Brassinosteroids (BRs), essential plant-specific steroidal hormones, function in a wide spectrum of plant growth and development events, including seed germination. Rice is not only a monocotyledonous model plant but also one of the most important staple food crops of human beings. Rice seed germination is a decisive event for the next-generation of plant growth and successful seed germination is critical for rice yield. However, little is known about the molecular mechanisms on how BR modulates seed germination in rice. In the present study, we used isobaric tags for relative and absolute quantification (iTRAQ) based proteomic approach to study BR-regulated proteome during the early stage of seed germination. The results showed that more than 800 BR-responsive proteins were identified, including 88 reliable target proteins responsive to stimuli of both BR-deficiency and BR-insensitivity. Moreover, 90% of the 88 target proteins shared a similar expression change pattern. Gene ontology and string analysis indicated that ribosomal structural proteins, as well as proteins involved in protein biosynthesis and carbohydrate metabolisms were highly clustered. These findings not only enrich BR-regulated protein database in rice seeds, but also allow us to gain novel insights into the molecular mechanism of BR regulated seed germination. PMID:27703189

  7. Type A acute aortic dissection in nonagenarian: rare but possible.

    PubMed

    Citro, Rodolfo; Patella, Marco Mariano; Provenza, Gennaro; Gregorio, Giovanni; Bossone, Eduardo

    2007-09-01

    Acute type A aortic dissection (TA-AAD) is a highly lethal clinical entity that can occur within a wide age range, associated with multiple aetiologies and various clinical presentations. In the very elderly type A aortic dissection frequently presents with non-specific symptoms and signs and is associated with high mortality and morbidity. Thus the clinician must have a high index of clinical suspicion in order to prompt the most appropriate diagnostic-therapeutic strategy. We report a nonagenarian women with TA-AAD, treated successfully with medical therapy.

  8. [Research progress of endoscopic submucosal dissection with traction].

    PubMed

    Wu, Wenming; Wei, Zhi; Sun, Ziqin

    2016-01-01

    Endoscopic submucosal dissection (ESD) is a widely accepted treatment for early gastrointestinal cancer. However, the steep learning curve of standard ESD procedures makes it less widely applied in clinical practice. Therefore, various traction devices and techniques have been applied previously to facilitate ESD, such as clip-with-line method, Spring-assisted method, S-O clip method, magnetic anchor method, and robot-assisted method. These methods create a clear field of vision and allow dissections to be performed more quickly and safely. In this article, we will discuss various techniques for achieving tractions during ESD on clinical decision, and their advantages and disadvantages. PMID:26797847

  9. Superior mesenteric artery dissection after extracorporeal shockwave lithotripsy.

    PubMed

    Bakoyiannis, Christos; Anastasiou, Ioannis; Koutsoumpelis, Andreas; Fragiadis, Evangelos; Felesaki, Eleni; Kafeza, Marina; Georgopoulos, Sotirios; Tsigris, Christos

    2012-01-01

    The use of shockwave lithotripsy is currently the mainstay of treatment in renal calculosis. Several complications including vessel injuries have been implied to extracorporeal shockwave lithotripsy. We report an isolated dissection of the superior mesenteric artery in a 60-year-old male presenting with abdominal pain which occurred three days after extracorporeal shockwave lithotripsy. The patient was treated conservatively and the abdominal pain subsided 24 hours later. The patient's history, the course of his disease, and the timing may suggest a correlation between the dissection and the ESWL.

  10. Dissecting thoracic aortic aneurysm associated with tuberculous pleural effusion

    PubMed Central

    Im, Kyong Shil; Choi, Min Kyung; Jeon, Yong Kyoung

    2016-01-01

    We present the case of thoracic aortic aneurysm associated with the tuberculous pleural effusion. An 82-year-old woman underwent emergency stent graft under a diagnosis of dissecting thoracic aortic aneurysm. Preoperative computed tomography revealed right pleural effusion supposed to the hemothorax caused by the dissecting aneurysm. But, the effusion was sanguineous color fluid and it was determined to result from pulmonary tuberculosis. The medical team was exposed to the pulmonary tuberculosis; fortunately no one became infected. Physicians should be aware of the possibility of an infected aortic aneurysm and prepare for pathogen transmission. PMID:27499987

  11. Basic dissecting techniques in ultrasound-guided surgery

    PubMed Central

    Koczy, Bogdan; Mielnik, Michał; Pilecki, Grzegorz; Dzielicki, Józef; Jakubowski, Wiesław

    2014-01-01

    Ultrasound-guided surgery is an area of minimally-invasive surgery where surgical procedures are performed with the aid of ultrasound imaging throghout the operation. This requires the operator to posses a certain degree of experience in endoscopic procedures, and to be adeptly skillfull in conducting US examinations. It is combining and finely tuning together these two elements that allows to perform efficiently an ultrasound-guided surgical procedure. Accessing an affected site correctly is of utmost importance in surgery, being oftentimes decisive in terms of the procedure's final outcome. In ultrasound-guided procedures, the operative site is accessed percutaneously, with a single point incision, yet tissues situated deeper within are dissected with dissecting techniques in a fluid evironment, typical for this area of surgery. Dissecting techniques in ultrasound-guided surgery are currently divided into basic ones which employ either a hydrodissection needle, surgical instruments, electrosurgical instruments, a thread, or a combination thereof, and advanced ones where either a balloon, a hook dissection technique, or a hybrid one is used. Hydrodissection with a needle was devised based on the rule of complementarity, and is the most frequently applied technique in ultrasound-guided surgery. The immense possibilities that go along with this modality will be of huge benefit to any surgeon, regardless of their field. Dissection with a variety of surgical instruments and electrosurgery instruments is a standard practice in all surgery areas, yet the method of imaging we employ in ultrasound-guided surgery results in certain modifications of these techniques. It is, however, learning the thread technique that facilitates a precise and oftentimes extensive dissection. This technique is successfully applied for dissecting muscle, ligament, tendon, vascular and other structures. Having mastered dissecting techniques allows to perform any minimally-invasive procedure

  12. Basic dissecting techniques in ultrasound-guided surgery.

    PubMed

    Pilecki, Zbigniew; Koczy, Bogdan; Mielnik, Michał; Pilecki, Grzegorz; Dzielicki, Józef; Jakubowski, Wiesław

    2014-06-01

    Ultrasound-guided surgery is an area of minimally-invasive surgery where surgical procedures are performed with the aid of ultrasound imaging throghout the operation. This requires the operator to posses a certain degree of experience in endoscopic procedures, and to be adeptly skillfull in conducting US examinations. It is combining and finely tuning together these two elements that allows to perform efficiently an ultrasound-guided surgical procedure. Accessing an affected site correctly is of utmost importance in surgery, being oftentimes decisive in terms of the procedure's final outcome. In ultrasound-guided procedures, the operative site is accessed percutaneously, with a single point incision, yet tissues situated deeper within are dissected with dissecting techniques in a fluid evironment, typical for this area of surgery. Dissecting techniques in ultrasound-guided surgery are currently divided into basic ones which employ either a hydrodissection needle, surgical instruments, electrosurgical instruments, a thread, or a combination thereof, and advanced ones where either a balloon, a hook dissection technique, or a hybrid one is used. Hydrodissection with a needle was devised based on the rule of complementarity, and is the most frequently applied technique in ultrasound-guided surgery. The immense possibilities that go along with this modality will be of huge benefit to any surgeon, regardless of their field. Dissection with a variety of surgical instruments and electrosurgery instruments is a standard practice in all surgery areas, yet the method of imaging we employ in ultrasound-guided surgery results in certain modifications of these techniques. It is, however, learning the thread technique that facilitates a precise and oftentimes extensive dissection. This technique is successfully applied for dissecting muscle, ligament, tendon, vascular and other structures. Having mastered dissecting techniques allows to perform any minimally-invasive procedure

  13. Spatial association between dissection density and environmental factors over the entire conterminous United States

    NASA Astrophysics Data System (ADS)

    Luo, Wei; Jasiewicz, Jaroslaw; Stepinski, Tomasz; Wang, Jinfeng; Xu, Chengdong; Cang, Xuezhi

    2016-01-01

    Previous studies of land dissection density (D) often find contradictory results regarding factors controlling its spatial variation. We hypothesize that the dominant controlling factors (and the interactions between them) vary from region to region due to differences in each region's local characteristics and geologic history. We test this hypothesis by applying a geographical detector method to eight physiographic divisions of the conterminous United States and identify the dominant factor(s) in each. The geographical detector method computes the power of determinant (q) that quantitatively measures the affinity between the factor considered and D. Results show that the factor (or factor combination) with the largest q value is different for physiographic regions with different characteristics and geologic histories. For example, lithology dominates in mountainous regions, curvature dominates in plains, and glaciation dominates in previously glaciated areas. The geographical detector method offers an objective framework for revealing factors controlling Earth surface processes.

  14. Snack Cake 'Dissection': A Flipped Classroom Exercise to Engage Undergraduates With Basic Neuroanatomy.

    PubMed

    Watson, Todd D

    2015-01-01

    This report describes a brief classroom activity for introducing basic neuroanatomical terminology and concepts to undergraduates in a survey-level course. Prior to completing the activity, students watched a short online lecture discussing the relevant material. During class, students worked in groups to 'dissect' snack cakes using the information they learned in the video and then reported and shared their 'results' using anatomical terminology. Quantitative feedback suggests that students found that the exercise was useful practice, that it helped increase their confidence in their ability to learn neuroanatomy, and that it helped increase their interest in the material generally. Data from an online anatomy quiz further supports the exercise's efficacy. Overall, the data suggest that this exercise may be a worthwhile addition to courses that introduce neuroanatomy to undergraduates.

  15. Adult-onset dysphagia lusoria secondary to a dissecting aberrant right subclavian artery associated with type B acute aortic dissection

    PubMed Central

    Singh, Sarabjeet; Grewal, Puneet Dhillon; Symons, John; Ahmed, Aziz; Khosla, Sandeep; Arora, Rohit

    2008-01-01

    The case of a 78-year-old African American woman who presented at the Mount Sinai Medical Center (Chicago, USA) with excruciating backache is presented. Computed tomography of the chest at the time of admission showed dissection of the aortic arch, descending aorta and dissection of an aberrant right subclavian artery. She was managed medically for Stanford type B acute aortic dissection. The patient was asymptomatic at presentation, but started complaining of new-onset dysphagia during her stay in the hospital. An esophagogram was performed and suggested posterior impingement of the esophagus, a classic sign of an aberrant right subclavian artery. Because the patient had multiple underlying comorbidities and the dysphagia was mild and intermittent, surgery was deferred. The patient was discharged home after complete stabilization and was scheduled for a follow-up appointment. PMID:18209773

  16. Role of cadaveric dissections in modern medical curricula: a study on student perceptions.

    PubMed

    Dissabandara, Lakal O; Nirthanan, Selvanayagam N; Khoo, Tien K; Tedman, Raymond

    2015-09-01

    The shift from traditional medical curricula to newer teaching and learning approaches such as problem-based learning has often resulted in omission or significant reduction of cadaveric dissections as a method of learning anatomy. The objective of this study was to evaluate students' perception of dissection in a graduate-entry, problem-based learning-based medical curriculum. At the end of the musculoskeletal dissection program in second year, a Likert-type questionnaire was used to explore medical student perceptions of the perceived advantages and challenges of cadaveric dissections in comparison with other anatomy teaching methods. Overall, a majority of students had a positive perception of dissections. Students who attended dissections regularly had significantly more positive perceptions about their experience and were in agreement with statements such as "dissections make learning more interesting" and "I would be disadvantaged if I did not attend dissection classes." Non-regular attendance was associated with statements about dissections such as "I do not like the smell," "time consuming," and "bored with the way it is carried-out." A follow-up study after completion of the medical program revealed a significant improvement of positive perception about dissection. Student perceptions appear to favour a role for cadaveric dissection in learning anatomy in modern medical curricula. However, optimal and effective integration of dissections is important, with consideration given to its structure and extent of content weighed against logistics and availability of resources; while addressing negative perceptions of dissection-based teaching.

  17. Role of cadaveric dissections in modern medical curricula: a study on student perceptions

    PubMed Central

    Nirthanan, Selvanayagam N.; Khoo, Tien K.; Tedman, Raymond

    2015-01-01

    The shift from traditional medical curricula to newer teaching and learning approaches such as problem-based learning has often resulted in omission or significant reduction of cadaveric dissections as a method of learning anatomy. The objective of this study was to evaluate students' perception of dissection in a graduate-entry, problem-based learning-based medical curriculum. At the end of the musculoskeletal dissection program in second year, a Likert-type questionnaire was used to explore medical student perceptions of the perceived advantages and challenges of cadaveric dissections in comparison with other anatomy teaching methods. Overall, a majority of students had a positive perception of dissections. Students who attended dissections regularly had significantly more positive perceptions about their experience and were in agreement with statements such as "dissections make learning more interesting" and "I would be disadvantaged if I did not attend dissection classes." Non-regular attendance was associated with statements about dissections such as "I do not like the smell," "time consuming," and "bored with the way it is carried-out." A follow-up study after completion of the medical program revealed a significant improvement of positive perception about dissection. Student perceptions appear to favour a role for cadaveric dissection in learning anatomy in modern medical curricula. However, optimal and effective integration of dissections is important, with consideration given to its structure and extent of content weighed against logistics and availability of resources; while addressing negative perceptions of dissection-based teaching. PMID:26417481

  18. Genetic dissection of a model complex trait using the Drosophila Synthetic Population Resource

    PubMed Central

    King, Elizabeth G.; Merkes, Chris M.; McNeil, Casey L.; Hoofer, Steven R.; Sen, Saunak; Broman, Karl W.; Long, Anthony D.; Macdonald, Stuart J.

    2012-01-01

    Genetic dissection of complex, polygenic trait variation is a key goal of medical and evolutionary genetics. Attempts to identify genetic variants underlying complex traits have been plagued by low mapping resolution in traditional linkage studies, and an inability to identify variants that cumulatively explain the bulk of standing genetic variation in genome-wide association studies (GWAS). Thus, much of the heritability remains unexplained for most complex traits. Here we describe a novel, freely available resource for the Drosophila community consisting of two sets of recombinant inbred lines (RILs), each derived from an advanced generation cross between a different set of eight highly inbred, completely resequenced founders. The Drosophila Synthetic Population Resource (DSPR) has been designed to combine the high mapping resolution offered by multiple generations of recombination, with the high statistical power afforded by a linkage-based design. Here, we detail the properties of the mapping panel of >1600 genotyped RILs, and provide an empirical demonstration of the utility of the approach by genetically dissecting alcohol dehydrogenase (ADH) enzyme activity. We confirm that a large fraction of the variation in this classic quantitative trait is due to allelic variation at the Adh locus, and additionally identify several previously unknown modest-effect trans-acting QTL (quantitative trait loci). Using a unique property of multiparental linkage mapping designs, for each QTL we highlight a relatively small set of candidate causative variants for follow-up work. The DSPR represents an important step toward the ultimate goal of a complete understanding of the genetics of complex traits in the Drosophila model system. PMID:22496517

  19. Proteomic and phosphoproteomic comparison of human ES and iPS cells.

    PubMed

    Phanstiel, Douglas H; Brumbaugh, Justin; Wenger, Craig D; Tian, Shulan; Probasco, Mitchell D; Bailey, Derek J; Swaney, Danielle L; Tervo, Mark A; Bolin, Jennifer M; Ruotti, Victor; Stewart, Ron; Thomson, James A; Coon, Joshua J

    2011-01-01

    Combining high-mass-accuracy mass spectrometry, isobaric tagging and software for multiplexed, large-scale protein quantification, we report deep proteomic coverage of four human embryonic stem cell and four induced pluripotent stem cell lines in biological triplicate. This 24-sample comparison resulted in a very large set of identified proteins and phosphorylation sites in pluripotent cells. The statistical analysis afforded by our approach revealed subtle but reproducible differences in protein expression and protein phosphorylation between embryonic stem cells and induced pluripotent cells. Merging these results with RNA-seq analysis data, we found functionally related differences across each tier of regulation. We also introduce the Stem Cell-Omics Repository (SCOR), a resource to collate and display quantitative information across multiple planes of measurement, including mRNA, protein and post-translational modifications.

  20. Aortic tear and dissection related to connective tissues abnormalities resembling Marfan syndrome in a Great Dane.

    PubMed

    Lenz, Jennifer A; Bach, Jonathan F; Bell, Cynthia M; Stepien, Rebecca L

    2015-06-01

    Aortic tears and acute aortic dissection are rarely reported in dogs. This report describes a case of aortic dissection and probable sinus of Valsalva rupture in a young Great Dane with associated histopathologic findings suggestive of a connective tissue abnormality.

  1. Multiple aneurism (Siraja Granthi) observed during cadaveric dissection

    PubMed Central

    Subbannavar, Pralhad D.

    2014-01-01

    Acharya Sushruta has emphasized the method and importance of dissection to study anatomy practically. Perfect knowledge of anatomy is vital for practicing surgeons and hence training of dissecting the dead body was considered as mandatory for surgeons. Though dissection techniques may give the perception of the structure of organs, the pervading and subtle consciousness in the body can be experienced with the eyes of knowledge and penance only. Though standard anatomy is defined based on statistical inferences on comparing large number of subjects, individual variations and exceptional structural specialties tend to occur quite frequently. Proper recording and publication of such instances would strengthen the knowledge base of the science. During the routine cadaveric dissection in the anatomy lab, multiple aneurisms (Siraja Granthi) in the abdominal aorta and femoral artery of 55-year-old male cadaver were observed. Such pathological variations are uncommon but clinically significant. Rupture of aneurysms or clot formation inside the lumen can produce serious complications in living condition. Enhancing size of the aneurysm producing pressure effect on the nearby structures can be the other reason for the surgical intervention. A good number of these can remain asymptomatic for a considerable period. As the clinical consequences are wide varying, the disease is of interest to physicians, as well as surgeons. PMID:25972721

  2. Multiple aneurism (Siraja Granthi) observed during cadaveric dissection.

    PubMed

    Subbannavar, Pralhad D

    2014-01-01

    Acharya Sushruta has emphasized the method and importance of dissection to study anatomy practically. Perfect knowledge of anatomy is vital for practicing surgeons and hence training of dissecting the dead body was considered as mandatory for surgeons. Though dissection techniques may give the perception of the structure of organs, the pervading and subtle consciousness in the body can be experienced with the eyes of knowledge and penance only. Though standard anatomy is defined based on statistical inferences on comparing large number of subjects, individual variations and exceptional structural specialties tend to occur quite frequently. Proper recording and publication of such instances would strengthen the knowledge base of the science. During the routine cadaveric dissection in the anatomy lab, multiple aneurisms (Siraja Granthi) in the abdominal aorta and femoral artery of 55-year-old male cadaver were observed. Such pathological variations are uncommon but clinically significant. Rupture of aneurysms or clot formation inside the lumen can produce serious complications in living condition. Enhancing size of the aneurysm producing pressure effect on the nearby structures can be the other reason for the surgical intervention. A good number of these can remain asymptomatic for a considerable period. As the clinical consequences are wide varying, the disease is of interest to physicians, as well as surgeons. PMID:25972721

  3. The Influence of Emotion on Students' Performance in Dissection Exercises

    ERIC Educational Resources Information Center

    Holstermann, Nina; Grube, Dietmar; Bogeholz, Susanne

    2009-01-01

    This paper investigates the issue of how emotions such as disgust influence students' self-efficacy belief in terms of mastering a dissection task and also how these affect their interest in the biology of the heart. Following models of intrinsic motivation and the development of motivation, we expected disgust to negatively impact on students'…

  4. Case report on vertebral artery dissection in mixed martial arts.

    PubMed

    Slowey, Michael; Maw, Graeme; Furyk, Jeremy

    2012-04-01

    A 41-year-old man presented to the ED with severe vertigo 2 days after a grappling injury while training in mixed martial arts. Imaging revealed a cerebellar infarct with complete occlusion of the right vertebral artery secondary to dissection. Management options are discussed as is the ongoing controversy regarding the safety of the sport. PMID:22487672

  5. There Is More to the Dissection of a Pig's Heart

    ERIC Educational Resources Information Center

    Lee, Yeung Chung

    2004-01-01

    The dissection of the mammalian heart in secondary biology classes need not be restricted to revealing the internal structure of the heart and its function. It could also be used to demonstrate other important aspects of blood circulation, including the blood supply to the heart itself as well as the causes and effects of coronary heart disease.…

  6. Standard methods for Apis mellifera anatomy and dissection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An understanding of the anatomy and functions of internal and external structures are fundamental to many studies on the honey bee Apis mellifera. Similarly, proficiency in dissection techniques is vital for many more complex procedures. In this paper, which is a prelude to the other papers of the C...

  7. Three cases of cervicocephalic artery dissection in an amusement park.

    PubMed

    Kurita, Naohide; Ueno, Yuji; Watanabe, Masao; Miyamoto, Nobukazu; Shimura, Hideki; Nonaka, Senshu; Tsutsumi, Satoshi; Yasumoto, Yukimasa; Hattori, Nobutaka; Urabe, Takao

    2014-01-01

    About 20 cases of cerebrovascular accidents in amusement parks have been documented. However, only a few cases of amusement park stroke (APS) after roller coaster rides have been reported. Here, we present triggers of stroke, clinical characteristics, and the angiographic appearance of 3 consecutive patients of APS. Their clinical characteristics included young age, absence of atherosclerotic risk factors, and severe injuries. Serial changes in angiographic appearance led to the diagnosis of cervicocephalic artery dissection (CAD). Patients A and B were diagnosed with isolated middle cerebral artery (MCA) dissection, and patient C was diagnosed with internal carotid artery dissection involving MCA dissection. Running excitedly toward an attraction in patient A, a go-kart ride in patient B, and riding in an enhanced motion vehicle in patient C were considered as the likely triggers for APS. We had specific cases with APS associated with CAD, which can occur under diverse contexts other than roller coaster rides at amusement parks. Our findings suggest that the variable directions of the high gravitational forces induced by vehicle riding or running excitedly might injure the MCA or internal carotid artery, and thereby cause CAD in the amusement park. PMID:25263648

  8. Case report on vertebral artery dissection in mixed martial arts.

    PubMed

    Slowey, Michael; Maw, Graeme; Furyk, Jeremy

    2012-04-01

    A 41-year-old man presented to the ED with severe vertigo 2 days after a grappling injury while training in mixed martial arts. Imaging revealed a cerebellar infarct with complete occlusion of the right vertebral artery secondary to dissection. Management options are discussed as is the ongoing controversy regarding the safety of the sport.

  9. Neurovascular Bundle Decompression without Excessive Dissection for Tarsal Tunnel Syndrome

    PubMed Central

    KIM, Kyongsong; ISU, Toyohiko; MORIMOTO, Daijiro; SASAMORI, Toru; SUGAWARA, Atsushi; CHIBA, Yasuhiro; ISOBE, Masahiro; KOBAYASHI, Shiro; MORITA, Akio

    2014-01-01

    Tarsal tunnel syndrome (TTS) is an entrapment neuropathy of the posterior tibial nerve and its branches in the tarsal tunnel. We present our less invasive surgical treatment of TTS in 69 patients (116 feet) and their clinical outcomes. The mean follow-up period was 64.6 months. With the patient under local anesthesia we use a microscope to perform sharp dissection of the flexor retinaculum and remove the connective tissues surrounding the posterior tibial nerve and vessels. To prevent postoperative adhesion and delayed neuropathy, decompression is performed to achieve symptom improvement without excessive dissection. Decompression is considered complete when the patient reports intraoperative symptom abatement and arterial pulsation is sufficient. The sensation of numbness and/or pain and of foreign substance adhesion was reduced in 92% and 95% of our patients, respectively. In self-assessments, 47 patients (68%) reported the treatment outcome as satisfactory, 15 (22%) as acceptable, and 7 (10%) were dissatisfied. Of 116 feet, 4 (3%) required re-operation, initial decompression was insufficient in 2 feet and further decompression was performed; in the other 2 feet improvement was achieved by decompression of the distal tarsal tunnel. Our surgical method involves neurovascular bundle decompression to obtain sufficient arterial pulsation. As we use local anesthesia, we can confirm symptom improvement intraoperatively, thereby avoiding unnecessary excessive dissection. Our method is simple, safe, and without detailed nerve dissection and it prevents postoperative adhesion. PMID:25367582

  10. Aneurysm Formation After Endovascular Treatment of Acute Type A Dissection.

    PubMed

    Yang, Lai; Wang, Jiaping

    2016-08-01

    Recently, reports have described successful endovascular stent graft (ESG) treatment of patients with acute type A aortic dissection. We report 1 ESG treatment for this condition and the complication of a new aneurysm formation during a 6-month follow-up. PMID:27630269

  11. Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

    PubMed Central

    Spratford, Carrie M.; Kumar, Justin P.

    2014-01-01

    A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations. PMID:25285379

  12. Dissecting cellulitis of the scalp: response to isotretinoin.

    PubMed

    Scerri, L; Williams, H C; Allen, B R

    1996-06-01

    We report three patients with dissecting cellulitis of the scalp. Prolonged treatment with oral isotretinoin was highly effective in all three patients. Furthermore, long-term post-treatment follow-up in two of the patients has shown a sustained therapeutic benefit. PMID:8763434

  13. Refractory dissecting Cellulitis of the Scalp Successfully controlled with Adalimumab.

    PubMed

    Martin-García, Rafael F; Rullán, Jennifer M

    2015-06-01

    Dissecting cellulitis of the scalp (DCS) is an uncommon inflammatory disease that often results in scarring alopecia. Numerous therapies have either proved ineffective or only temporarily effective in the management of this condition. Recent reports show adequate responses to tumor necrosis factor (TNF) inhibitors in cases of DCS. We report a case of severe recalcitrant DCS successfully treated with adalimumab. PMID:26061062

  14. Dissecting cellulitis of the scalp in 2 girls.

    PubMed

    Ramesh, V

    1990-01-01

    Two girls with dissecting cellulitis of the scalp are described. In one Pseudomonas aeruginosa was isolated from the sinus discharge. Both patients were controlled with prolonged antibiotic therapy and periodic aspiration of the fluctuant lesions. Role of infection in perpetuating the condition is highlighted. PMID:2106459

  15. Three cases of cervicocephalic artery dissection in an amusement park.

    PubMed

    Kurita, Naohide; Ueno, Yuji; Watanabe, Masao; Miyamoto, Nobukazu; Shimura, Hideki; Nonaka, Senshu; Tsutsumi, Satoshi; Yasumoto, Yukimasa; Hattori, Nobutaka; Urabe, Takao

    2014-01-01

    About 20 cases of cerebrovascular accidents in amusement parks have been documented. However, only a few cases of amusement park stroke (APS) after roller coaster rides have been reported. Here, we present triggers of stroke, clinical characteristics, and the angiographic appearance of 3 consecutive patients of APS. Their clinical characteristics included young age, absence of atherosclerotic risk factors, and severe injuries. Serial changes in angiographic appearance led to the diagnosis of cervicocephalic artery dissection (CAD). Patients A and B were diagnosed with isolated middle cerebral artery (MCA) dissection, and patient C was diagnosed with internal carotid artery dissection involving MCA dissection. Running excitedly toward an attraction in patient A, a go-kart ride in patient B, and riding in an enhanced motion vehicle in patient C were considered as the likely triggers for APS. We had specific cases with APS associated with CAD, which can occur under diverse contexts other than roller coaster rides at amusement parks. Our findings suggest that the variable directions of the high gravitational forces induced by vehicle riding or running excitedly might injure the MCA or internal carotid artery, and thereby cause CAD in the amusement park.

  16. Using a Dissecting Microscope in Teaching Introductory Chemistry.

    ERIC Educational Resources Information Center

    Winokur, Robert; Monroe, Manus

    1985-01-01

    To have students develop observational skills and acquire an excitement about chemistry, stereoscopic dissecting microscopes are used to observe the physical characteristics and chemical reactions of various substances. Several of these reactions (including dissolving potassium permanganate in deionized water and reactions between copper metal and…

  17. Coronary Artery Dissection: Not Just a Heart Attack

    MedlinePlus

    ... Stroke More Coronary Artery Dissection: Not Just a Heart Attack Updated:May 24,2016 Sometimes a heart attack ... Disease Go Red For Women Types of aneurysms Heart Attack • Home • About Heart Attacks Acute Coronary Syndrome (ACS) • ...

  18. SUBMUCOSAL DISSECTION OF THE RETROPHARYNGEAL SPACE DURING NASAL INTUBATION.

    PubMed

    Hakim, Mumin; Cartabuke, Richard S; Krishna, Senthil G; Veneziano, Giorgio; Syed, Ahsan; Lind, Meredith N Merz; Tobias, Qoseph D

    2015-10-01

    Various complications have been reported with nasal endotracheal intubation including bleeding, epistaxis, bacteremia, damage to intranasal structures, and even intracranial penetration. We present two cases that required general anesthesia for dental surgery. Submucosal dissection of the retropharyngeal tissues occurred during attempted nasal endotracheal intubation. Previous reports of this complication are reviewed, treatment strategies presented, and potential maneuvers to prevent this complication suggested. PMID:26860021

  19. A Novel Approach to the Dissection of the Human Knee

    ERIC Educational Resources Information Center

    Clemente, F. Richard; Fabrizio, Philip A.; Shumaker, Michael

    2009-01-01

    The knee is one of the most frequently injured joints of the human body with injuries affecting the general population and the athletic population of many age groups. Dissection procedures for the knee joint typically do not allow unobstructed visualization of the anterior cruciate or posterior cruciate ligaments without sacrificing the collateral…

  20. Refining the laparoscopic retroperitoneal lymph node dissection for testicular cancer.

    PubMed

    Romero, Frederico R; Wagner, Andrew; Brito, Fabio A; Muntener, Michael; Lima, Guilherme C; Kavoussi, Louis R

    2006-01-01

    Since its initial description, the laparoscopic retroperitoneal lymph node dissection has evolved considerably, from a purely diagnostic tool performed to stage germ cell testicular cancer to a therapeutic operation that fully duplicates the open technique. Herein, we describe the current technique employed at our institution, along with illustrations of all surgical steps, and delineate the refinements of the technique over time.

  1. Student Attitudes to Whole Body Donation Are Influenced by Dissection

    ERIC Educational Resources Information Center

    Cahill, Kevin C.; Ettarh, Raj R.

    2008-01-01

    Given the important role that anatomical dissection plays in the shaping of medical student attitudes to life and death, these attitudes have not been evaluated in the context of whole body donation for medical science. First year students of anatomy in an Irish university medical school were surveyed by questionnaire before and after the initial…

  2. [Spontaneous renal artery dissection with renal infarction: a case report].

    PubMed

    Oki, Takashi; Adachi, Hiroyuki; Tahara, Hideo; Kino, Sigeo

    2011-11-01

    A 58-year-old woman visited our hospital with nausea and right flank pain. At first abdominal ultrasonography was performed, suggesting a right renal infarction. Computed tomography (CT) study of the abdomen with intravenous contrast was performed to determine the cause of the symptoms. The scan revealed poor enhancement in the lower half of the right kidney. She was diagnosed with a right renal infarction. She was initially treated with anticoagulant therapy, but 5 days later, she complained of nausea. This time, CT demonstrated exacerbation of a right renal infarction with renal artery dissection. Based on this finding, we performed a right nephrectomy. The result of pathology was segmental arterial mediolysis. She was discharged 12 days after the surgery and is doing well at 6 months after discharge. Spontaneous renal artery dissection is a rare disease. It constitutes approximately 0.05% of arteriographic dissections. In addition, spontaneous renal artery dissection shows nonspecific symptoms. Together, these two factors may cause a delay in diagnosis.

  3. General Dissection of Female Ant Reproductive System and Brain

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dissection of the reproductive system of ant workers and queens can be useful for answering many questions. Observations of ovarian status in both female castes can be used to identify relationships between other factors and the ovaries, determine whether an individual has laid eggs, and, with more ...

  4. Dissecting microtubule structures by laser ablation.

    PubMed

    Decker, Franziska; Brugués, Jan

    2015-01-01

    Here, we describe a detailed protocol, based on laser ablation and fluorescence optical microscopy, to measure the microtubule organization in spindles, including microtubule length distribution, polarity, and plus and minus end densities. The method uses the asymmetry in microtubule depolymerization after a cut, where the newly created microtubule plus ends depolymerize all the way to the minus ends, whereas the newly created minus ends remain stable. The protocol described in this chapter is optimized for spindles, but can be easily applied to any microtubule-based structure. The chapter is divided into two parts. First, we provide the theoretical basis for the method. Second, we describe in detail all steps necessary to reconstruct the microtubule organization of a spindle assembled in Xenopus laevis egg extract. Compared to electron microscopy, which in theory can resolve individual microtubules in spindles and provide similar structural information, our method is fast and simple enough to allow for a full quantitative reconstruction of the microtubule organization of several X. laevis spindles—which have volumes tens of thousands of times larger than spindles whose structures have been previously solved by electron microscopy—in a single experimental session, as well as to explore how the architecture of these structures changes in response to biochemical perturbations.

  5. Relative quantification of phosphoproteomic changes in grapevine (Vitis vinifera L.) leaves in response to abscisic acid

    PubMed Central

    Rattanakan, Supakan; George, Iniga; Haynes, Paul A; Cramer, Grant R

    2016-01-01

    In a previous transcriptomic analysis, abscisic acid (ABA) was found to affect the abundance of a number of transcripts in leaves of Cabernet Sauvignon grapevines with roots that had been exposed to 10 μm ABA for 2 h. Other work has indicated that ABA affects protein abundance and protein phosphorylation as well. In this study we investigated changes in protein abundance and phosphorylation of Cabernet Sauvignon grapevine leaves. Protein abundance was assessed by both label-free and isobaric-label quantitive proteomic methods. Each identified common proteins, but also additional proteins not found with the other method. Overall, several thousand proteins were identified and several hundred were quantified. In addition, hundreds of phosphoproteins were identified. Tens of proteins were found to be affected in the leaf after the roots had been exposed to ABA for 2 h, more than half of them were phosphorylated proteins. Many phosphosites were confirmed and several new ones were identified. ABA increased the abundance of some proteins, but the majority of the proteins had their protein abundance decreased. Many of these proteins were involved in growth and plant organ development, including proteins involved in protein synthesis, photosynthesis, sugar and amino-acid metabolism. This study provides new insights into how ABA regulates plant responses and acclimation to water deficits. PMID:27366326

  6. Activating Mutations in PIK3CA Lead to Widespread Modulation of the Tyrosine Phosphoproteome

    PubMed Central

    Blair, Brian G.; Pinto, Sneha M.; Nirujogi, Raja S.; Jelinek, Christine A.; Malhotra, Radhika; Kim, Min-Sik; Park, Ben Ho; Pandey, Akhilesh

    2015-01-01

    The human oncogene PIK3CA is frequently mutated in human cancers. Two hotspot mutations in PIK3CA, E545K and H1047R, have been shown to regulate widespread signaling events downstream of AKT, leading to increased cell proliferation, growth, survival, and motility. We used quantitative mass spectrometry to profile the global phosphotyrosine proteome of isogenic knock-in cell lines containing these activating mutations, where we identified 824 unique phosphopeptides. Although it is well understood that these mutations result in hyperactivation of the serine/threonine kinase AKT, we found a surprisingly widespread modulation of tyrosine phosphorylation levels of proteins in the mutant cells. In the tyrosine kinome alone, 29 tyrosine kinases were altered in their phosphorylation status. Many of the regulated phosphosites that we identified were located in the kinase domain or the canonical activation sites, indicating that these kinases and their downstream signaling pathways were activated. Our study demonstrates that there is frequent and unexpected cross-talk that occurs between tyrosine signaling pathways and serine/threonine signaling pathways activated by the canonical PI3K-AKT axis. PMID:26267517

  7. A Comparison of V-Frog[C] to Physical Frog Dissection

    ERIC Educational Resources Information Center

    Lalley, James P.; Piotrowski, Phillip S.; Battaglia, Barbara; Brophy, Keith; Chugh, Kevin

    2010-01-01

    The purpose of the present study was to examine and compare the effectiveness of virtual frog dissection using V-Frog[C] and physical frog dissection on learning, retention, and affect. Subjects were secondary students enrolled in year-long life science classes in a suburban high school (N=102). Virtual dissections were done with V-Frog[C], a…

  8. Perceived Disgust and Personal Experiences are Associated with Acceptance of Dissections in Schools

    ERIC Educational Resources Information Center

    Fancovicova, Jana; Prokop, Pavol; Leskova, Andrea

    2013-01-01

    Animal dissections are essential parts of anatomy/zoology courses, but their effectiveness is influenced by student attitudes and emotions. Here we examined attitudes toward dissections in 397 prospective biology teachers enrolling two Slovak universities. Perceived disgust of dissections negatively correlated with other attitudes toward…

  9. The First Cut Is the Deepest: Reflections on the State of Animal Dissection in Biology Education

    ERIC Educational Resources Information Center

    De Villiers, Rian; Monk, Martin

    2005-01-01

    In biology education, the study of structure has traditionally involved the use of dissection. Animal-rights campaigners have caused biology educators and learners to question the necessity of dissections. This study reviews the research evidence for the efficacy of alternatives to dissection and then turns to research evidence on attitudes to…

  10. An Investigative Alternative to Single-Species Dissection in the Introductory Biology Laboratory

    ERIC Educational Resources Information Center

    Carlin, Joel L.

    2010-01-01

    Dissections of single species (e.g., fetal pig) are a common student learning activity in introductory biology courses. Such dissections demonstrate location of anatomical parts and provide dissection practice but provide less opportunity for student critical thinking, numeracy and demonstration of the scientific method. A comparative anatomy lab…

  11. Biology Teachers' Dissection Practices and the Influences that Lead to Their Adoption: An Exploratory Research

    ERIC Educational Resources Information Center

    Milano, Regina Nicole

    2010-01-01

    The lack of resolution in the on-going animal dissection debate inspired this mixed methods study to identify Connecticut secondary biology teachers' dissection practices and the influences that lead to their adoption. Qualitative findings indicate past experiences, managing objections to dissection, school culture, goals of biology teaching and…

  12. [Skin collagen abnormalities in a Japanese patient with extracranial internal carotid artery dissection followed by extracranial vertebral artery dissection].

    PubMed

    Sengoku, Renpei; Sato, Hironori; Honda, Hidehiko; Inoue, Kiyoharu; Ono, Seiitsu

    2006-02-01

    A 41-year-old man with hypertension and hyperlipidemia who complained of left hemiparesis after a temporal headache was admitted to our hospital. A cervical MRI with gadolinium enhancement revealed an intramural hematoma is compatible with right extracranial internal carotid artery dissection. Two weeks later, he complained of sudden onset of pain in the right side of his neck. The right extracranial internal carotid artery dissection followed by the right extracranial vertebral artery dissection was diagnosed. Spontaneous cervical artery dissection (SCAD) is one of the causes of stroke in young adults. The pathogenesis of SCAD remains unknown. Minor trauma like an excessive sneeze, migraine, and connective tissue disorders such as fibromuscular dysplasia and Ehlers-Danlos syndrome are well-known as risk factors for SCAD. Pathologically skin collagen abnormalities have been seen in German patients with SCAD without clinical evidence for any specific connective tissue disorder. We examined the ultrastructural morphology of the Japanese patient's dermal connective tissue components by electron microscopy. The patient's collagen fibers contained fibrils with highly variable diameters, and there were other ultrastructural abnormalities, including flower-like fibrils and large-diameter composite fibrils. This is the first report of a case of ultrastructural abnormalities of dermal connective tissue in a Japanese patient with SCAD. PMID:16619839

  13. Analysis of dissected tissues with digital holographic microscopy: quantification of inflammation mediated tissue alteration, influence of sample preparation, and reliability of numerical autofocusing

    NASA Astrophysics Data System (ADS)

    Kemper, Björn; Lenz, Philipp; Bettenworth, Dominik; Krausewitz, Philipp; Domagk, Dirk; Ketelhut, Steffi

    2015-03-01

    Quantitative phase imaging with digital holographic microscopy (DHM) allows label-free imaging of tissue sections and quantification of the spatial refractive index distribution, which is of interest for applications in digital pathology. We show that DHM allows quantitative imaging of different layers in unstained tissue samples by detection of refractive index changes. In addition, we evaluate the automated refocussing feature of DHM for application on dissected tissues and could achieve highly reproducible holographic autofocusing for unstained and moderately stained samples. Finally, it is demonstrated that in human ulcerative colitis patients the average tissue refractive index is reduced significantly in all parts of the inflamed colonic wall in comparison to patients in remission.

  14. Genome-wide association mapping of quantitative resistance to sudden death syndrome in soybean

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sudden death syndrome (SDS) is a serious threat to soybean production that can be controlled by host plant resistance. To dissect the genetic architecture of quantitative resistance to the disease in soybean, two independent association panels of soybean elite cultivar, consisting of 392 and 300 uni...

  15. Mapping quantitative trait loci for plant adaptation and morphology traits in wheat using single nucleotide polymorphisms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wheat (Triticum aestivum L.) morphological and adaptation-related traits that are controlled by quantitative traits loci (QTL) define potential growing areas of a wheat cultivar. To dissect the QTL for heading date (HD), lodging resistance (LR), shattering resistance (SR), cold tolerance (CT), plant...

  16. Secondary science classroom dissections: Informing policy by evaluating cognitive outcomes and exploring affective outcomes

    NASA Astrophysics Data System (ADS)

    Allspaw, Kathleen M.

    Animal protection organizations claim that dissection is pedagogically unsound and that it will cause students to lose respect for non-human animals. Science teacher organizations support curricula that teach respect for animal life and include dissection. Prior research compared dissection to dissection alternatives. Four of the six studies revealed no difference between groups on tests of cognitive outcomes. One study revealed that dissection was superior, and one revealed that the alternative was superior. No differences in attitudes toward science, dissection or school were found. Attitudes toward non-human animals were not measured. This study focused on the dissections of earthworms and frogs in middle and high school classrooms. Pre and post-tests of conceptual understanding revealed failing scores and no significant pre/post differences. Because these tests required critical thinking skills, and the dissection activities did not, it is difficult to determine if the poor performance on these tests indicates the inability of the students to think critically, and/or if it indicates the ineffectiveness of dissection. Further studies of dissections that focus on critical thinking would be necessary to make this distinction. Classroom observations, student written narratives, and student and adult interviews revealed mixed attitudes toward non-human animals. Student behaviors during dissection were similar to those behaviors exhibited during non-dissection activities. Most students and adults readily supported worm dissections while they expressed some trepidation about frog dissections. Students and adults universally expressed affection for their pets and opposed the use of their own pets for dissection/research. There was slight support for the use of dogs and cats for dissection/research, but only those students who expressed hate for cats said that they could dissect cats. None of the students or adults expressed a willingness to dissect dogs. Some students

  17. Principles for Management of Intraoperative Acute Type A Aortic Dissection.

    PubMed

    Gukop, Philemon; Chandrasekaran, Vankatachalam

    2015-12-01

    Intraoperative Type A aortic dissection is a rare pathology with incidence of 0.06-0.32%. It is associated with a high mortality between 30-50%. Some associated risk factors, including hypertension, enlarged aorta, peripheral vascular disease, advanced age, atheroma, and high arterial pressure on cardiopulmonary bypass, have been identified. Modification of these risk factors could reduce the incidence of this event. Prompt diagnosis and management, with the aid of intraoperative trans-esophageal echocardiography and/or epi-aortic ultrasound has been shown to reduce the mortality to 17%. We illustrate the principles of management of this pathology with the case of a 62-year-old female who developed acute Type A aortic dissection while undergoing minimally invasive mitral valve repair.

  18. Iatrogenic dissection of the descending aorta: Conservative or endovascular treatment?

    PubMed Central

    Baikoussis, Nikolaos G.; Argiriou, Michalis; Kratimenos, Theodoros; Karameri, Vasiliki; Dedeilias, Panagiotis

    2016-01-01

    Transcatheter aortic valve implantation (TAVI) is without any doubt a standard technique and the treatment of choice of severe aortic valve stenosis (AVS) in very high-operative risk patients. However, a number of complications may occur and has been described. Improper valve position, valve migration, paravalvular regurgitation, conduction disturbances, stroke and aortic dissection have been succeeded despite the perfection of the technique. For anyone of the complications above described, a solution may be invented. We present an interesting case of an 81-year-old woman with severe AVS treated through TAVI due to very high operative risk. This female, 12 days later presented with thoracic pain and shortness of breath and through the computed tomography of the chest performed was diagnosed a dissection of the descending aorta. She successfully underwent on thoracic endovascular aortic repair. In this report, we refer the bibliographic data and we discuss the treatment options in these cases. PMID:27397470

  19. Femtosecond laser dissection in C. elegans neural circuits

    NASA Astrophysics Data System (ADS)

    Samuel, Aravinthan D. T.; Chung, Samuel H.; Clark, Damon A.; Gabel, Christopher V.; Chang, Chieh; Murthy, Venkatesh; Mazur, Eric

    2006-02-01

    The nematode C. elegans, a millimeter-long roundworm, is a well-established model organism for studies of neural development and behavior, however physiological methods to manipulate and monitor the activity of its neural network have lagged behind the development of powerful methods in genetics and molecular biology. The small size and transparency of C. elegans make the worm an ideal test-bed for the development of physiological methods derived from optics and microscopy. We present the development and application of a new physiological tool: femtosecond laser dissection, which allows us to selectively ablate segments of individual neural fibers within live C. elegans. Femtosecond laser dissection provides a scalpel with submicrometer resolution, and we discuss its application in studies of neural growth, regenerative growth, and the neural basis of behavior.

  20. [Stanford type a acute aortic dissection with pectus excavatum].

    PubMed

    Kuwata, Toshiyuki; Fukuda, Hirotsugu; Yoshitatsu, Masao; Yamada, Yasuyuki; Shibasaki, Ikuko; Inoue, Yuho; Hori, Takayuki; Ogawa, Hironaga; Tsuchiya, Go; Shimizu, Riha; Takei, Yusuke

    2012-11-01

    Pectus excavatum is generally an isolated abnormality of the chest wall. However, some patients have a concomitant pectus deformity and cardiac & aortic disease. Decisions must be made regarding the operative approach and whether the pectus excavatum should be corrected during the same session. We report 2 patients with acute Stanford type A aortic dissection and pectus excavatum who underwent emergency operation. In case 1, median sternotomy is an unsuitable approach for open heart surgery, since the heart and great vessels are displace into the left hemithorax. But combined sternotomy and left anterior thoracotomy provided excellent surgical exposure. In case 2, we proceeded with a leftsided costotomy of four ribs and place a normal chest retractor providing as excellent exposure as combined sternotomy and left anterior thoracotomy. A left-sided costotomy of four ribs can be performed safely, eliminating the risks of median sternotomy in acute stanford type A aortic dissection with pectus excavatum.